Simian Adenoviruses SAdV-36, -42.1, -42.2, and -44 and Uses Thereof

ABSTRACT

A recombinant vector comprises simian adenovirus 36, simian adenovirus 42.1, simian adenovirus 42.2 and/or simian adenovirus 44 sequences and a heterologous gene under the control of regulatory sequences. A cell line which expresses one or more simian adenovirus-36, -42.1, -42.2 or -44 gene(s) is also described. Methods of using the vectors and cell lines are provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.P30-DK47757 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Adenovirus is a double-stranded DNA virus with a genome size of about 36kilobases (kb), which has been widely used for gene transferapplications due to its ability to achieve highly efficient genetransfer in a variety of target tissues and large transgene capacity.Conventionally, E1 genes of adenovirus are deleted and replaced with atransgene cassette consisting of the promoter of choice, cDNA sequenceof the gene of interest and a poly A signal, resulting in a replicationdefective recombinant virus.

Adenoviruses have a characteristic morphology with an icosahedral capsidconsisting of three major proteins, hexon (II), penton base (III) and aknobbed fibre (IV), along with a number of other minor proteins, VI,VIII, IX, IIIa and IVa2 [W. C. Russell, J. Gen Virol., 81:2573-2604(November 2000)]. The virus genome is a linear, double-stranded DNA witha terminal protein attached covalently to the 5′ terminus, which haveinverted terminal repeats (ITRs). The virus DNA is intimately associatedwith the highly basic protein VII and a small peptide pX (formerlytermed mu). Another protein, V, is packaged with this DNA-proteincomplex and provides a structural link to the capsid via protein VI. Thevirus also contains a virus-encoded protease, which is necessary forprocessing of some of the structural proteins to produce matureinfectious virus.

A classification scheme has been developed for the Mastadenovirusfamily, which includes human, simian, bovine, equine, porcine, ovine,canine and opossum adenoviruses. This classification scheme wasdeveloped based on the differing abilities of the adenovirus sequencesin the family to agglutinate red blood cells. The result was sixsubgroups, now referred to as subgroups A, B, C, D, E and F. See, T.Shenk et al., Adenoviridae: The Viruses and their Replication”, Ch. 67,in FIELD'S VIROLOGY, 6^(th) Ed., edited by B. N Fields et al,(Lippincott Raven Publishers, Philadelphia, 1996), p. 111-2112.

Recombinant adenoviruses have been described for delivery ofheterologous molecules to host cells. See, U.S. Pat. No. 6,083,716,which describes the genome of two chimpanzee adenoviruses Simianadenoviruses, C5, C6 and C7, have been described in U.S. Pat. No.7,247,472 as being useful as vaccine vectors. Other chimpanzeeadenoviruses are described in WO 2005/1071093 as being useful for makingadenovirus vaccine carriers.

What is needed in the art are effective vectors which avoid the effectof pre-existing immunity to selected adenovirus serotypes in thepopulation.

SUMMARY OF THE INVENTION

Isolated nucleic acid sequences and amino acid sequences of simianadenovirus 36 (SAdV-36), simian adenovirus 42.1 (SAdV-42.1), simianadenovirus 42.2 (SAdV-42.2), simian adenovirus 44 (SAdV-44), and vectorscontaining these sequences are provided herein. Also provided are anumber of methods for using the vectors and cells of the invention.

The methods described herein involve delivering one or more selectedheterologous gene(s) to a mammalian patient by administering a vector ofthe invention. Use of the compositions described herein for vaccinationpermits presentation of a selected antigen for the elicitation ofprotective immune responses. The vectors based on SAdV-36, SAdV-42.1,SAdV-42.2 and SAdV-44 may also be used for producing heterologous geneproducts in vitro. Such gene products are themselves useful in a varietyfor a variety of purposes such as are described herein.

These and other embodiments and advantages of the invention aredescribed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the polyfunctionality of CD8+ T cells in response tostimulation with a influenza nucleoprotein peptide following vaccinationwith C36 CMV PI FluA NP (a recombinant SAdV-36 virus harboring the NPexpression cassette—see Example 4)) and AdH5-FluA NP (a recombinantHAdV-5 virus harboring the NP expression cassette), as described inExample 5. For both SAdV-36 and HAdV-5, the percentage of cytokine+ CD8+T cells is shown. In each bar, the top segment reflects the subset ofCD8+ T cells secreting IFN-γ, but not IL2 or TNFα. The middle segmentreflects the subset of CD8+ T cells secreting IFN-γ and TNFα, but notIL2. The bottom segment reflects the subset of CD8+ T cells secretingIFN-γ, IL2, and TNFα.

DETAILED DESCRIPTION OF THE INVENTION

Novel nucleic acid and amino acid sequences from simian adenovirus 36(SAdV-36), SAdV-42.1, SAdV-42.2 and SAdV-44, which was isolated fromsimian feces, are provided. Also provided are novel adenovirus vectorsand packaging cell lines to produce those vectors for use in the invitro production of recombinant proteins or fragments or other reagents.Further provided are compositions for use in delivering a heterologousmolecule for therapeutic or vaccine purposes. Such therapeutic orvaccine compositions contain the adenoviral vectors carrying an insertedheterologous molecule. In addition, the novel SAdV-36, SAdV-42.1,SAdV-42.2 and SAdV-44 sequences are useful in providing the essentialhelper functions required for production of recombinant adeno-associatedviral (AAV) vectors. Thus, helper constructs, methods and cell lineswhich use these sequences in such production methods, are provided.

SAdV-36 has been determined by the inventors to be in the same subgroupas human subgroup E adenoviruses. Since this virus is from subgroup E,the capsid of this virus (optionally an intact or recombinant viralparticle or an empty capsid) is useful in method of inducing animmunomodulatory effect or enhanced immune response by delivering anadenovirus subgroup E capsid to subject. The SAdV-36 capsid can bedelivered alone or in a combination regimen with an active agent toenhance the immune response thereto. In another aspect, a method ofinducing interferon alpha production in a subject in need thereofcomprising delivering the SAdV-36 capsid to a subject is provided. Instill another aspect, a method for producing one or more cytokines inculture is provided. This method involves incubating a culturecontaining dendritic cells and the SAdV-36 capsid under conditionssuitable to produce cytokines/chemokines, including, among others, alphainterferon.

SAdV-42.1, 42.2 and 44 have been determined by the inventors to bewithin the same subgroup as human subgroup C adenoviruses. SAdV-42.1 andSAdV-42.2 were previously identified to be within the same phylogenicsubgroup as human subgroup C adenoviruses. SAdV-42.1 and SAdV-42.2 werepreviously identified as SAdV-42 and SAdV-43 in US Provisional PatentApplication Nos. 61/068,069 (filed Mar. 4, 2008) and 61/067,993 (filedMar. 4, 2008), respectively, from which priority is claimed. While theseadenoviruses may not be serologically distinct, their revisednomenclature reflects that they are closely structurally related,differing by approximately six amino acids or fewer within theirrespective hexon regions.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or aprotein thereof, or a fragment thereof which is at least 8 amino acids,or more desirably, at least 15 amino acids in length. Examples ofsuitable fragments are described herein.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length of the genome(e.g., about 36 kbp), the full-length of an open reading frame of agene, protein, subunit, or enzyme [see, e.g., the tables providing theadenoviral coding regions], or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Similarly, “percent sequenceidentity” may be readily determined for amino acid sequences, over thefull-length of a protein, or a fragment thereof. Suitably, a fragment isat least about 8 amino acids in length, and may be up to about 700 aminoacids. Examples of suitable fragments are described herein.

Identity is readily determined using such algorithms and computerprograms as are defined herein at default settings. Preferably, suchidentity is over the full length of the protein, enzyme, subunit, orover a fragment of at least about 8 amino acids in length. However,identity may be based upon shorter regions, where suited to the use towhich the identical gene product is being put.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as “Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI® utilities [InVitrogen] are also used. Thereare also a number of algorithms known in the art that can be used tomeasure nucleotide sequence identity, including those contained in theprograms described above. As another example, polynucleotide sequencescan be compared using Fasta, a program in GCG Version 6.1. Fastaprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences. For instance,percent sequence identity between nucleic acid sequences can bedetermined using Fasta with its default parameters (a word size of 6 andthe NOPAM factor for the scoring matrix) as provided in GCG Version 6.1,herein incorporated by reference. Similarly programs are available forperforming amino acid alignments. Generally, these programs are used atdefault settings, although one of skill in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program that provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs.

“Recombinant”, as applied to a polynucleotide, means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. A site-specificrecombination site that has been cloned into a genome of a virus orviral vector, wherein the genome of the virus does not naturally containit, is a heterologous recombination site. When a polynucleotide with anencoding sequence for a recombinase is used to genetically alter a cellthat does not normally express the recombinase, both the polynucleotideand the recombinase are heterologous to the cell.

As used throughout this specification and the claims, the term“comprise” and its variants including, “comprises”, “comprising”, amongother variants, is inclusive of other components, elements, integers,steps and the like. The term “consists of” or “consisting of” areexclusive of other components, elements, integers, steps and the like.

I. The Simian Adenovirus Sequences

The invention provides nucleic acid sequences and amino acid sequencesof simian adenovirus 36 (SAdV-36), SAdV-42.1, SAdV42.2, and SAdV-44,each of which are isolated from the other material with which they areassociated in nature.

A. Nucleic Acid Sequences

The SAdV-36 nucleic acid sequences provided herein include nucleotides 1to 36556 of SEQ ID NO:1. The SAdV-42.1 nucleic acid sequences providedherein include nucleotides 1 to 37786 of SEQ ID NO:33. The SAdV-42.2nucleic acid sequences provided herein include nucleotides 1 to 37820 ofSEQ ID NO:64. The SAdV-44 nucleic acid sequences provided herein includenucleotides 1 to 37711 of SEQ ID NO:95. See Sequence Listing, which isincorporated by reference herein.

In one embodiment, the nucleic acid sequences of the invention furtherencompass the strand which is complementary to the sequences of SEQ IDNO: 1, 33, 64 and 95, as well as the RNA and cDNA sequencescorresponding to the sequences of the following sequences and theircomplementary strands. In another embodiment, the nucleic acid sequencesfurther encompass sequences which are greater than 98.5% identical, andpreferably, greater than about 99% identical, to the Sequence Listing.Also included in one embodiment, are natural variants and engineeredmodifications of the sequences provided in SEQ ID NO: 1, 33, 64 and 95,and their complementary strands. Such modifications include, forexample, labels that are known in the art, methylation, and substitutionof one or more of the naturally occurring nucleotides with a degeneratenucleotide.

TABLE 1 NUCLEIC ACID REGIONS SAdV-36 SAdV-42.1 SAdV-42.2 SAdV-44 ORF ORFORF ORF SEQ ID SEQ ID SEQ ID SEQ ID Regions NO: 1 NO: 33 NO: 64 NO: 95ITR 1 . . . 124 1 . . . 109 1 . . . 119 1 . . . 106 E1a Join Join JoinJoin 576 . . . 1143, 566 . . . 1106, 576 . . . 1116, 576 . . . 1116,1228 . . . 1433 1217 . . . 1512 1229 . . . 1524 1226 . . . 1521 E1bSmall 1598 . . . 2173 1597 . . . 2247 1609 . . . 2259 1606 . . . 2256T/19K Large 1903 . . . 3414 1989 . . . 3512 2001 . . . 3524 1998 . . .3521 T/55K IX 3502 . . . 3927 3612 . . . 4052 3624 . . . 4064 3621 . . .4061 E2b pTP Complement Complement Complement Complement (8458 . . .10380, (8627 . . . 10618, (8639 . . . 10642, (8636 . . . 10639, 13827 .. . 13835) 14208 . . . 14216) 14235 . . . 14243) 14231 . . . 14239)Polymerase Complement Complement Complement Complement (5096 . . . 8656,(5223 . . . 8825, (5235 . . . 8837, (5232 . . . 8834, 13827 . . . 13835)14208 . . . 14216) 14235 . . . 14243) 14231 . . . 14239) IVa2 ComplementComplement Complement Complement (3993 . . . 5323, (4117 . . . 5450,(4129 . . . 5462, (4126 . . . 5459, 5602 . . . 5614) 5729 . . . 5741)5741 . . . 5753) 5738 . . . 5750) L1 52/55D 10815 . . . 11996 11097 . .. 12356 11117 . . . 12376 11114 . . . 12370 IIIa 12023 . . . 13798 12383. . . 14152 12403 . . . 14172 12397 . . . 14166 L2 Penton 13880 . . .15502 14258 . . . 16000 14285 . . . 16042 14281 . . . 16023 VII 15509 .. . 16093 16006 . . . 16602 16048 . . . 16644 16029 . . . 16625 V 16141. . . 17175 16696 . . . 17817 16743 . . . 17843 16724 . . . 17815 pX17201 . . . 17431 17845 . . . 18084 17872 . . . 18111 17844 . . . 18083L3 VI 17507 . . . 18229 18191 . . . 18967 18219 . . . 18995 18190 . . .18966 Hexon 18324 . . . 21146 19098 . . . 21968 19126 . . . 21196 19097. . . 21982 Endo- 21173 . . . 21793 22001 . . . 22633 22029 . . . 2266122015 . . . 22647 protease E2a DBP Complement Complement ComplementComplement (21881 . . . 23416) (22764 . . . 24404) (22792 . . . 24423)(22773 . . . 24407) L4 100 kD 23439 . . . 25841 24442 . . . 26934 24461. . . 26950 24445 . . . 26940 33 kD Join Join Join Join homolog 25552 .. . 25888, 26633 . . . 26973, 26649 . . . 26989, 26639 . . . 26969,26058 . . . 26392 27293 . . . 27569 27315 . . . 27591 27187 . . . 2757522 kD 25552 . . . 26121 26633 . . . 27253 26649 . . . 27275 26639 . . .27259 VIII 26467 . . . 27147 27648 . . . 28328 27670 . . . 28350 27654 .. . 28334 E3 12.5K 27151 . . . 27468 28332 . . . 28646 28354 . . . 2866828338 . . . 28652 CR1- 27425 . . . 28048 28603 . . . 29157 28625 . . .29179 28657 . . . 29163 alpha gp19K 28033 . . . 28566 29344 . . . 2982029366 . . . 29842 29350 . . . 29826 CR1- 28600 . . . 29217 29852 . . .30751 29874 . . . 30782 29858 . . . 30766 beta CR1- 29233 . . . 2984430751 . . . 31560 30782 . . . 31588 30766 . . . 31581 gamma CR1- 29862 .. . 30743 — — — delta RID- 30754 . . . 31026 31575 . . . 31844 31603 . .. 31872 31596 . . . 31865 alpha RID-beta 31035 . . . 31466 31850 . . .32248 31878 . . . 32276 31871 . . . 32269 14.7K 31462 . . . 31866 32244. . . 32627 32272 . . . 32655 32265 . . . 32648 L5 Fiber 32167 . . .33441 32836 . . . 34566 32810 . . . 34594 32803 . . . 34482 E4 Orf 6/7Complement Complement Complement Complement (33557 . . . 33808, (34755 .. . 35027, (34782 . . . 35054, (34672 . . . 34944, 34540 . . . 34710)35730 . . . 35912) 35757 . . . 35939) 35647 . . . 35829) Orf 6Complement Complement Complement Complement (33808 . . . 34710) (35031 .. . 35912) (35058 . . . 35939) (34948 . . . 35829) Orf 4 ComplementComplement Complement Complement (34619 . . . 34981) (35815 . . . 36180)(35842 . . . 36207) (35732 . . . 36097) Orf 3 Complement ComplementComplement Complement (34994 . . . 35344) (36190 . . . 36534) (36217 . .. 36561) (36107 . . . 36451) Orf 2 Complement Complement ComplementComplement (35344 . . . 35730) (36534 . . . 36923) (36561 . . . 36950)(36451 . . . 36840) Orf1 Complement Complement Complement Complement(35773 . . . 36144) (36987 . . . 37370) (37011 . . . 37394) (36902 . . .37285) ITR Complement Complement Complement Complement (36433 . . .36556) (37678 . . . 37786) (37702 . . . 37820) (37606 . . . 37711)

In one embodiment, fragments of the sequences of SAdV-36, SAdV-42.1,SAdV-42.2 and SAdV-44, and their complementary strand, cDNA and RNAcomplementary thereto are provided. Suitable fragments are at least 15nucleotides in length, and encompass functional fragments, i.e.,fragments which are of biological interest. For example, a functionalfragment can express a desired adenoviral product or may be useful inproduction of recombinant viral vectors. Such fragments include the genesequences and fragments listed in the tables herein. The tables providethe transcript regions and open reading frames in the SAdV-36,SAdV-42.1, SAdV-42.2 and SAdV-44 sequences. For certain genes, thetranscripts and open reading frames (ORFs) are located on the strandcomplementary to that presented in SEQ ID NO:1, 33, 64 and 95. See,e.g., E2b, E4 and E2a. The calculated molecular weights of the encodedproteins are also shown. Note that the E1a open reading frame ofSAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 and the E2b open readingframe contain internal splice sites. These splice sites are noted in thetable above.

The SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 adenoviral nucleic acidsequences are useful as therapeutic agents and in construction of avariety of vector systems and host cells. As used herein, a vectorincludes any suitable nucleic acid molecule including, naked DNA, aplasmid, a virus, a cosmid, or an episome. These sequences and productsmay be used alone or in combination with other adenoviral sequences orfragments, or in combination with elements from other adenoviral ornon-adenoviral sequences. The SAdV-36, SAdV-42.1, SAdV-42.2 and/orSAdV-44 sequences are also useful as antisense delivery vectors, genetherapy vectors, or vaccine vectors. Thus, further provided are nucleicacid molecules, gene delivery vectors, and host cells which contain theSAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 sequences.

For example, the invention encompasses a nucleic acid moleculecontaining simian Ad ITR sequences of the invention. In another example,the invention provides a nucleic acid molecule containing simian Adsequences of the invention encoding a desired Ad gene product. Stillother nucleic acid molecule constructed using the sequences of theinvention will be readily apparent to one of skill in the art, in viewof the information provided herein.

In one embodiment, the simian Ad gene regions identified herein may beused in a variety of vectors for delivery of a heterologous molecule toa cell. For example, vectors are generated for expression of anadenoviral capsid protein (or fragment thereof) for purposes ofgenerating a viral vector in a packaging host cell. Such vectors may bedesigned for expression in trans. Alternatively, such vectors aredesigned to provide cells which stably contain sequences which expressdesired adenoviral functions, e.g., one or more of E1a, E1b, theterminal repeat sequences, E2a, E2b, E4, E4ORF6 region.

In addition, the adenoviral gene sequences and fragments thereof areuseful for providing the helper functions necessary for production ofhelper-dependent viruses (e.g., adenoviral vectors deleted of essentialfunctions, or adeno-associated viruses (AAV)). For such productionmethods, the SAdV-36, SAdV-42.1, SAdV-42.2 and SAdV-44 sequences can beutilized in such a method in a manner similar to those described for thehuman Ad. However, due to the differences in sequences between theSAdV-36, SAdV-42.1, SAdV-42.2 and SAdV-44 sequences and those of humanAd, the use of the SAdV-36, SAdV-42.1, SAdV-42.2 and SAdV-44 sequencesgreatly minimize or eliminate the possibility of homologousrecombination with helper functions in a host cell carrying human Ad E1functions, e.g., 293 cells, which may produce infectious adenoviralcontaminants during rAAV production.

Methods of producing rAAV using adenoviral helper functions have beendescribed at length in the literature with human adenoviral serotypes.See, e.g., U.S. Pat. No. 6,258,595 and the references cited therein.See, also, U.S. Pat. No. 5,871,982; WO 99/14354; WO 99/15685; WO99/47691. These methods may also be used in production of non-humanserotype AAV, including non-human primate AAV serotypes. The SAdV-36,SAdV-42.1, SAdV-42.2 and SAdV-44 sequences which provide the necessaryhelper functions (e.g., E1a, E1b, E2a and/or E4 ORF6) can beparticularly useful in providing the necessary adenoviral function whileminimizing or eliminating the possibility of recombination with anyother adenoviruses present in the rAAV-packaging cell which aretypically of human origin. Thus, selected genes or open reading framesof the SAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44 sequences may beutilized in these rAAV production methods.

Alternatively, recombinant SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44vectors may be utilized in these methods. Such recombinant adenoviralsimian vectors may include, e.g., a hybrid chimp Ad/AAV in which chimpAd sequences flank a rAAV expression cassette composed of, e.g., AAV 3′and/or 5′ ITRs and a transgene under the control of regulatory sequenceswhich control its expression. One of skill in the art will recognizethat still other simian adenoviral vectors and/or SAdV-36, SAdV-42.1,SAdV-42.2 and/or SAdV-44 gene sequences will be useful for production ofrAAV and other viruses dependent upon adenoviral helper.

In still another embodiment, nucleic acid molecules are designed fordelivery and expression of selected adenoviral gene products in a hostcell to achieve a desired physiologic effect. For example, a nucleicacid molecule containing sequences encoding an SAdV-36, SAdV-42.1,SAdV-42.2 and/or SAdV-44 E1a protein may be delivered to a subject foruse as a cancer therapeutic. Optionally, such a molecule is formulatedin a lipid-based carrier and preferentially targets cancer cells. Such aformulation may be combined with other cancer therapeutics (e.g.,cisplatin, taxol, or the like). Still other uses for the adenoviralsequences provided herein will be readily apparent to one of skill inthe art.

In addition, one of skill in the art will readily understand that theSAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 sequences can be readilyadapted for use for a variety of viral and non-viral vector systems forin vitro, ex vivo or in vivo delivery of therapeutic and immunogenicmolecules. For example, the SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44simian Ad sequences can be utilized in a variety of rAd and non-rAdvector systems. Such vectors systems may include, e.g., plasmids,lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadeno-associated viral systems, among others. Selection of these vectorsystems is not a limitation of the present invention.

The invention further provides molecules useful for production of thesimian and simian-derived proteins of the invention. Such moleculeswhich carry polynucleotides including the simian Ad DNA sequences of theinvention can be in the form of naked DNA, a plasmid, a virus or anyother genetic element.

B. SAdV-36, SAdV-42.1, SAdV-42.2 and SAdV-44 Adenoviral Proteins

Gene products of the SAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44adenovirus, such as proteins, enzymes, and fragments thereof, which areencoded by the adenoviral nucleic acids described herein are provided.Further encompassed are SAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44proteins, enzymes, and fragments thereof, having the amino acidsequences encoded by these nucleic acid sequences which are generated byother methods. Such proteins include those encoded by the open readingframes identified in Table 1, above, the proteins in Table 2, below,(also shown in the Sequence Listing) and fragments thereof of theproteins and polypeptides.

TABLE 2 PROTEIN SEQUENCES SAdV-44 SAdV-36 SAdV-42.1 SAdV-42.2 SEQRegions SEQ ID NO: SEQ ID NO: SEQ ID NO: ID NO: E1a 32 61 92 123 E1bSmall 24 54 65 96 T/19K Large 2 34 85 116 T/55K IX 3 35 66 97 L1 52/55D4 36 67 98 IIIa 5 37 68 99 L2 Penton 6 38 69 100 VII 7 39 70 101 V 8 4071 102 pX 9 41 72 103 L3 VI 10 42 73 104 Hexon 11 43 74 105 Endo 12 4475 106 protease L4 100 kD 13 45 76 107 33 kD 30 63 94 125 homolog 22 kD26 56 87 118 VIII 14 46 77 108 E3 12.5k 15 57 78 109 CR1- 27 47 88 110alpha gp19K 16 48 79 111 CR1-beta 17 49 80 112 CR1- 18 58 89 119 gammaCR1- 19 — — — delta RID- 20 50 81 113 alpha RID-beta 28 51 82 114 14.7 K21 59 90 120 L5 Fiber 22 52 83 121

Thus, in one aspect, unique simian adenoviral 36 (SAdV-36), SAdV-42.1,SAdV-42.2 or SAdV-44 proteins which are substantially pure, i.e., arefree of other viral and proteinaceous proteins are provided. Preferably,these proteins are at least 10% homogeneous, more preferably 60%homogeneous, and most preferably 95% homogeneous.

In one embodiment, unique simian-derived capsid proteins are provided.As used herein, a simian-derived capsid protein includes any adenoviralcapsid protein that contains a SAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44capsid protein or a fragment thereof, as defined above, including,without limitation, chimeric capsid proteins, fusion proteins,artificial capsid proteins, synthetic capsid proteins, and recombinantcapsid proteins, without limitation to means of generating theseproteins.

Suitably, these simian-derived capsid proteins contain one or moreSAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44 regions or fragments thereof(e.g., a hexon, penton, fiber, or fragment thereof) in combination withcapsid regions or fragments thereof of different adenoviral serotypes,or modified simian capsid proteins or fragments, as described herein. A“modification of a capsid protein associated with altered tropism” asused herein includes an altered capsid protein, i.e, a penton, hexon orfiber protein region, or fragment thereof, such as the knob domain ofthe fiber region, or a polynucleotide encoding same, such thatspecificity is altered. The simian-derived capsid may be constructedwith one or more of the simian Ad of the invention or another Adserotype which may be of human or non-human origin. Such Ad may beobtained from a variety of sources including the ATCC, commercial andacademic sources, or the sequences of the Ad may be obtained fromGenBank or other suitable sources.

The amino acid sequences of the penton protein of SAdV-36 (SEQ ID NO:6),SAdV-42.1 (SEQ ID NO:38), SAdV-42.2 (SEQ ID NO:69) and SAdV-44 (SEQ IDNO: 100) are provided. Suitably, this penton protein, or uniquefragments thereof, may be utilized for a variety of purposes. Examplesof suitable fragments include the penton having N-terminal and/orC-terminal truncations of about 50, 100, 150, or 200 amino acids, basedupon the amino acid numbering provided above and in SEQ ID NO:6, 38, 69and 100. Other suitable fragments include shorter internal, C-terminal,or N-terminal fragments. Further, the penton protein may be modified fora variety of purposes known to those of skill in the art.

Also provided are the amino acid sequences of the hexon protein ofSAdV-36 (SEQ ID NO:11), SAdV-42.1 (SEQ ID NO:43), SAdV-42.2 (SEQ IDNO:74) and SAdV-44 (SEQ ID NO: 105). Suitably, this hexon protein, orunique fragments thereof, may be utilized for a variety of purposes.Examples of suitable fragments include the hexon having N-terminaland/or C-terminal truncations of about 50, 100, 150, 200, 300, 400, or500 amino acids, based upon the amino acid numbering provided above andin SEQ ID NO: 11, 43, 74 or 105. Other suitable fragments includeshorter internal, C-terminal, or N-terminal fragments. For example, onesuitable fragment the loop region (domain) of the hexon protein,designated DE1 and FG1, or a hypervariable region thereof. Suchfragments include the regions spanning amino acid residues about 125 to443; about 138 to 441, or smaller fragments, such as those spanningabout residue 138 to residue 163; about 170 to about 176; about 195 toabout 203; about 233 to about 246; about 253 to about 264; about 287 toabout 297; and about 404 to about 430 of the simian hexon proteins, withreference to SEQ ID NO: 11, 43, 74 or 105. Other suitable fragments maybe readily identified by one of skill in the art. Further, the hexonprotein may be modified for a variety of purposes known to those ofskill in the art. Because the hexon protein is the determinant forserotype of an adenovirus, such artificial hexon proteins would resultin adenoviruses having artificial serotypes. Other artificial capsidproteins can also be constructed using the chimp Ad penton sequencesand/or fiber sequences of the invention and/or fragments thereof.

In one embodiment, an adenovirus having an altered hexon proteinutilizing the sequences of a SAdV-36, SAdV-42.1, SAdV-42.2 and/orSAdV-44 hexon protein may be generated. One suitable method for alteringhexon proteins is described in U.S. Pat. No. 5,922,315, which isincorporated by reference. In this method, at least one loop region ofthe adenovirus hexon is changed with at least one loop region of anotheradenovirus serotype. Thus, at least one loop region of such an alteredadenovirus hexon protein is a simian Ad hexon loop region of SAdV-36,SAdV-42.1, SAdV-42.2 or SAdV-44. In one embodiment, a loop region of theSAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44 hexon protein is replaced by aloop region from another adenovirus serotype. In another embodiment, theloop region of the SAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44 hexon isused to replace a loop region from another adenovirus serotype. Suitableadenovirus serotypes may be readily selected from among human andnon-human serotypes, as described herein. The selection of a suitableserotype is not a limitation of the present invention. Still other usesfor the SAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44 hexon protein sequenceswill be readily apparent to those of skill in the art.

The amino acid sequences of the fiber proteins of SAdV-36 (SEQ IDNO:22), SAdV-42.1 (SEQ ID NO:52), SAdV-42.2 (SEQ ID NO:83) and SAdV-44(SEQ ID NO:121) are also provided. Suitably, these fiber proteins, orunique fragments thereof, may be utilized for a variety of purposes. Onesuitable fragment is the fiber knob, located within SEQ ID NO: 22, 52,83 or 121. Examples of other suitable fragments include the fiber havingN-terminal and/or C-terminal truncations of about 50, 100, 150, or 200amino acids, based upon the amino acid numbering provided in SEQ ID NO:22, 52, 83 or 121. Still other suitable fragments include internalfragments. Further, the fiber protein may be modified using a variety oftechniques known to those of skill in the art.

Unique fragments of the proteins of the SAdV-36, SAdV-42.1, SAdV-42.2and/or SAdV-44 are at least 8 amino acids in length. However, fragmentsof other desired lengths can be readily utilized. In addition,modifications as may be introduced to enhance yield and/or expression ofa SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 gene product, e.g.,construction of a fusion molecule in which all or a fragment of theSAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 gene product is fused(either directly or via a linker) with a fusion partner to enhance areprovided herein. Other suitable modifications include, withoutlimitation, truncation of a coding region (e.g., a protein or enzyme) toeliminate a pre- or pro-protein ordinarily cleaved and to provide themature protein or enzyme and/or mutation of a coding region to provide asecretable gene product. Still other modifications will be readilyapparent to one of skill in the art. Further encompassed are proteinshaving at least about 99% identity to the SAdV-36, SAdV-42.1, SAdV-42.2and/or SAdV-44 proteins provided herein.

As described herein, vectors of the invention containing the adenoviralcapsid proteins of SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 areparticularly well suited for use in applications in which theneutralizing antibodies diminish the effectiveness of other Ad serotypebased vectors, as well as other viral vectors. The rAd vectors areparticularly advantageous in readministration for repeat gene therapy orfor boosting immune response (vaccine titers).

Under certain circumstances, it may be desirable to use one or more ofthe SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 gene products (e.g., acapsid protein or a fragment thereof) to generate an antibody. The term“an antibody,” as used herein, refers to an immunoglobulin moleculewhich is able to specifically bind to an epitope. The antibodies mayexist in a variety of forms including, for example, high affinitypolyclonal antibodies, monoclonal antibodies, synthetic antibodies,chimeric antibodies, recombinant antibodies and humanized antibodies.Such antibodies originate from immunoglobulin classes IgG, IgM, IgA, IgDand IgE.

Such antibodies may be generated using any of a number of methods knowin the art. Suitable antibodies may be generated by well-knownconventional techniques, e.g., Kohler and Milstein and the many knownmodifications thereof. Similarly desirable high titer antibodies aregenerated by applying known recombinant techniques to the monoclonal orpolyclonal antibodies developed to these antigens [see, e.g., PCT PatentApplication No. PCT/GB85/00392; British Patent Application PublicationNo. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al.,1989 Proc. Nat'l. Acad. Sci. USA, 86:10029-10033; PCT Patent ApplicationNo. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988);Huse et al, 1988a Science, 246:1275-1281]. Alternatively, antibodies canbe produced by manipulating the complementarity determining regions ofanimal or human antibodies to the antigen of this invention. See, e.g.,E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4,The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology ofMonoclonal Antibodies, Springer-Verlag (June, 1994); Harlow et al.,1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; and Bird et al., 1988, Science 242:423-426.Further provided by the present invention are anti-idiotype antibodies(Ab2) and anti-anti-idiotype antibodies (Ab3). See, e.g., M. Wettendorffet al., “Modulation of anti-tumor immunity by anti-idiotypicantibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J.Hiernaux, 1990 J. Am. Soc. Microbiol., Washington D.C.: pp. 203-229].These anti-idiotype and anti-anti-idiotype antibodies are produced usingtechniques well known to those of skill in the art. These antibodies maybe used for a variety of purposes, including diagnostic and clinicalmethods and kits.

Under certain circumstances, it may be desirable to introduce adetectable label or a tag onto a SAdV-36, SAdV-42.1, SAdV-42.2 and/orSAdV-44 gene product, antibody or other construct of the invention. Asused herein, a detectable label is a molecule which is capable, alone orupon interaction with another molecule, of providing a detectablesignal. Most desirably, the label is detectable visually, e.g. byfluorescence, for ready use in immunohistochemical analyses orimmunofluorescent microscopy. For example, suitable labels includefluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin(APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), andPE-Texas Red (ECD). All of these fluorescent dyes are commerciallyavailable, and their uses known to the art. Other useful labels includea colloidal gold label. Still other useful labels include radioactivecompounds or elements. Additionally, labels include a variety of enzymesystems that operate to reveal a colorimetric signal in an assay, e.g.,glucose oxidase (which uses glucose as a substrate) releases peroxide asa product which in the presence of peroxidase and a hydrogen donor suchas tetramethyl benzidine (TMB) produces an oxidized TMB that is seen asa blue color. Other examples include horseradish peroxidase (HRP),alkaline phosphatase (AP), and hexokinase in conjunction withglucose-6-phosphate dehydrogenase which reacts with ATP, glucose, andNAD+ to yield, among other products, NADH that is detected as increasedabsorbance at 340 nm wavelength.

Other label systems that are utilized in the methods described hereinare detectable by other means, e.g., colored latex microparticles [BangsLaboratories, Indiana] in which a dye is embedded are used in place ofenzymes to form conjugates with the target sequences provide a visualsignal indicative of the presence of the resulting complex in applicableassays.

Methods for coupling or associating the label with a desired moleculeare similarly conventional and known to those of skill in the art. Knownmethods of label attachment are described [see, for example, Handbook ofFluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland,Molecular Probes, Inc., Eugene, Oreg., 1996; Pierce Catalog andHandbook, Life Science and Analytical Research Products, Pierce ChemicalCompany, Rockford, Ill., 1994/1995]. Thus, selection of the label andcoupling methods do not limit this invention.

The sequences, proteins, and fragments of SAdV-36, SAdV-42.1, SAdV-42.2and/or SAdV-44 may be produced by any suitable means, includingrecombinant production, chemical synthesis, or other synthetic means.Suitable production techniques are well known to those of skill in theart. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively,peptides can also be synthesized by the well known solid phase peptidesynthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962);Stewart and Young, Solid Phase Peptide Synthesis (Freeman, SanFrancisco, 1969) pp. 27-62). These and other suitable production methodsare within the knowledge of those of skill in the art and are not alimitation of the present invention.

In addition, one of skill in the art will readily understand that theSAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 sequences can be readilyadapted for use for a variety of viral and non-viral vector systems forin vitro, ex vivo or in vivo delivery of therapeutic and immunogenicmolecules. For example, in one embodiment, the simian Ad capsid proteinsand other simian adenovirus proteins described herein are used fornon-viral, protein-based delivery of genes, proteins, and otherdesirable diagnostic, therapeutic and immunogenic molecules. In one suchembodiment, a protein of the invention is linked, directly orindirectly, to a molecule for targeting to cells with a receptor foradenoviruses. Preferably, a capsid protein such as a hexon, penton,fiber or a fragment thereof having a ligand for a cell surface receptoris selected for such targeting. Suitable molecules for delivery areselected from among the therapeutic molecules described herein and theirgene products. A variety of linkers including, lipids, polyLys, and thelike may be utilized as linkers. For example, the simian penton proteinmay be readily utilized for such a purpose by production of a fusionprotein using the simian penton sequences in a manner analogous to thatdescribed in Medina-Kauwe L K, et al, Gene Ther. 2001 May; 8(10):795-803and Medina-Kauwe L K, et al, Gene Ther. 2001 December; 8(23): 1753-1761.Alternatively, the amino acid sequences of simian Ad protein IX may beutilized for targeting vectors to a cell surface receptor, as describedin US Patent Appln 20010047081. Suitable ligands include a CD40 antigen,an RGD-containing or polylysine-containing sequence, and the like. Stillother simian Ad proteins, including, e.g., the hexon protein and/or thefiber protein, may be used for used for these and similar purposes.

Still other SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 adenoviralproteins may be used as alone, or in combination with other adenoviralprotein, for a variety of purposes which will be readily apparent to oneof skill in the art. In addition, still other uses for the SAdV-36,SAdV-42.1, SAdV-42.2 and/or SAdV-44 adenoviral proteins will be readilyapparent to one of skill in the art.

II. Recombinant Adenoviral Vectors

The compositions described herein include vectors that deliver aheterologous molecule to cells, either for therapeutic or vaccinepurposes. As used herein, a vector may include any genetic elementincluding, without limitation, naked DNA, a phage, transposon, cosmid,episome, plasmid, or a virus. Such vectors contain simian adenovirus DNAof SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44, and a minigene. By“minigene” is meant the combination of a selected heterologous gene andthe other regulatory elements necessary to drive translation,transcription and/or expression of the gene product in a host cell.

Typically, a SAdV-derived adenoviral vector is designed such that theminigene is located in a nucleic acid molecule which contains otheradenoviral sequences in the region native to a selected adenoviral gene.The minigene may be inserted into an existing gene region to disrupt thefunction of that region, if desired. Alternatively, the minigene may beinserted into the site of a partially or fully deleted adenoviral gene.For example, the minigene may be located in the site of such as the siteof a functional E1 deletion or functional E3 deletion, among others thatmay be selected. The term “functionally deleted” or “functionaldeletion” means that a sufficient amount of the gene region is removedor otherwise damaged, e.g., by mutation or modification, so that thegene region is no longer capable of producing functional products ofgene expression. If desired, the entire gene region may be removed.Other suitable sites for gene disruption or deletion are discussedelsewhere in the application.

For example, for a production vector useful for generation of arecombinant virus, the vector may contain the minigene and either the 5′end of the adenoviral genome or the 3′ end of the adenoviral genome, orboth the 5′ and 3′ ends of the adenoviral genome. The 5′ end of theadenoviral genome contains the 5′ cis-elements necessary for packagingand replication; i.e., the 5′ inverted terminal repeat (ITR) sequences(which function as origins of replication) and the native 5′ packagingenhancer domains (that contain sequences necessary for packaging linearAd genomes and enhancer elements for the E1 promoter). The 3′ end of theadenoviral genome includes the 3′ cis-elements (including the ITRs)necessary for packaging and encapsidation. Suitably, a recombinantadenovirus contains both 5′ and 3′ adenoviral cis-elements and theminigene is located between the 5′ and 3′ adenoviral sequences. ASAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 based adenoviral vector mayalso contain additional adenoviral sequences.

Suitably, these SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 basedadenoviral vectors contain one or more adenoviral elements derived fromthe adenoviral genome of the invention. In one embodiment, the vectorscontain adenoviral ITRs from SAdV-36, SAdV-42.1, SAdV-42.2 and/orSAdV-44 and additional adenoviral sequences from the same adenoviralserotype. In another embodiment, the vectors contain adenoviralsequences that are derived from a different adenoviral serotype thanthat which provides the ITRs.

As defined herein, a pseudotyped adenovirus refers to an adenovirus inwhich the capsid protein of the adenovirus is from a differentadenovirus than the adenovirus which provides the ITRs.

Further, chimeric or hybrid adenoviruses may be constructed using theadenoviruses described herein using techniques known to those of skillin the art. See, e.g., U.S. Pat. No. 7,291,498.

The selection of the adenoviral source of the ITRs and the source of anyother adenoviral sequences present in vector is not a limitation of thepresent embodiment. A variety of adenovirus strains are available fromthe American Type Culture Collection, Manassas, Va., or available byrequest from a variety of commercial and institutional sources. Further,the sequences of many such strains are available from a variety ofdatabases including, e.g., PubMed and GenBank. Homologous adenovirusvectors prepared from other simian or from human adenoviruses aredescribed in the published literature [see, for example, U.S. Pat. No.5,240,846]. The DNA sequences of a number of adenovirus types areavailable from GenBank, including type Ad5 [GenBank Accession No.M73260]. The adenovirus sequences may be obtained from any knownadenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, andfurther including any of the presently identified human types. Similarlyadenoviruses known to infect non-human animals (e.g., simians) may alsobe employed in the vector constructs of this invention. See, e.g., U.S.Pat. No. 6,083,716.

The viral sequences, helper viruses (if needed), and recombinant viralparticles, and other vector components and sequences employed in theconstruction of the vectors described herein are obtained as describedabove. The SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 DNA sequences ofthe invention are employed to construct vectors and cell lines useful inthe preparation of such vectors.

Modifications of the nucleic acid sequences forming the vectors of thisinvention, including sequence deletions, insertions, and other mutationsmay be generated using standard molecular biological techniques and arewithin the scope of this embodiment.

A. The “Minigene”

The methods employed for the selection of the transgene, the cloning andconstruction of the “minigene” and its insertion into the viral vectorare within the skill in the art given the teachings provided herein.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins comprising a membrane boundprotein appropriately fused to an antigen tag domain from, among others,hemagglutinin or Myc. These coding sequences, when associated withregulatory elements which drive their expression, provide signalsdetectable by conventional means, including enzymatic, radiographic,colorimetric, fluorescence or other spectrographic assays, fluorescentactivating cell sorting assays and immunological assays, includingenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for beta-galactosidase activity. Where the transgene is GFP orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

In one embodiment, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA moleculesinclude tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs.One example of a useful RNA sequence is a sequence which extinguishesexpression of a targeted nucleic acid sequence in the treated animal.

The transgene may be used for treatment, e.g., of genetic deficiencies,as a cancer therapeutic or vaccine, for induction of an immune response,and/or for prophylactic vaccine purposes. As used herein, induction ofan immune response refers to the ability of a molecule (e.g., a geneproduct) to induce a T cell and/or a humoral immune response to themolecule. The invention further includes using multiple transgenes,e.g., to correct or ameliorate a condition caused by a multi-subunitprotein. In certain situations, a different transgene may be used toencode each subunit of a protein, or to encode different peptides orproteins. This is desirable when the size of the DNA encoding theprotein subunit is large, e.g., for an immunoglobulin, theplatelet-derived growth factor, or a dystrophin protein. In order forthe cell to produce the multi-subunit protein, a cell is infected withthe recombinant virus containing each of the different subunits.Alternatively, different subunits of a protein may be encoded by thesame transgene. In this case, a single transgene includes the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribozyme entry site (IRES). This is desirable when thesize of the DNA encoding each of the subunits is small, e.g., the totalsize of the DNA encoding the subunits and the IRES is less than fivekilobases. As an alternative to an IRES, the DNA may be separated bysequences encoding a 2A peptide, which self-cleaves in apost-translational event. See, e.g., M. L. Donnelly, et al, J. Gen.Virol., 78(Pt 1):13-21 (January 1997); Furler, S., et al, Gene Ther.,8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817(May 2001). This 2A peptide is significantly smaller than an IRES,making it well suited for use when space is a limiting factor. However,the selected transgene may encode any biologically active product orother product, e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis embodiment.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements necessary which areoperably linked to the transgene in a manner that permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product.

A great number of expression control sequences, including promoterswhich are native, constitutive, inducible and/or tissue-specific, areknown in the art and may be utilized. Examples of constitutive promotersinclude, without limitation, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art. Forexample, inducible promoters include the zinc-inducible sheepmetallothionine (MT) promoter and the dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter. Other inducible systemsinclude the T7 polymerase promoter system [WO 98/10088]; the ecdysoneinsect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351(1996)], the tetracycline-repressible system [Gossen et al, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system[Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al,Curr. Opin. Chem. Biol., 2:512-518 (1998)]. Other systems include theFK506 dimer, VP16 or p65 using castradiol, diphenol murislerone, theRU486-inducible system [Wang et al, Nat. Biotech., 15:239-243 (1997) andWang et al, Gene Ther., 4:432-441 (1997)] and the rapamycin-induciblesystem [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)]. Theeffectiveness of some inducible promoters increases over time. In suchcases one can enhance the effectiveness of such systems by insertingmultiple repressors in tandem, e.g., TetR linked to a TetR by an IRES.Alternatively, one can wait at least 3 days before screening for thedesired function. One can enhance expression of desired proteins byknown means to enhance the effectiveness of this system. For example,using the Woodchuck Hepatitis Virus Posttranscriptional RegulatoryElement (WPRE).

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal β-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally occurringpromoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bonesialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and theneuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)),among others.

Optionally, vectors carrying transgenes encoding therapeutically usefulor immunogenic products may also include selectable markers or reportergenes may include sequences encoding geneticin, hygromicin or purimycinresistance, among others. Such selectable reporters or marker genes(preferably located outside the viral genome to be packaged into a viralparticle) can be used to signal the presence of the plasmids inbacterial cells, such as ampicillin resistance. Other components of thevector may include an origin of replication. Selection of these andother promoters and vector elements are conventional and many suchsequences are available [see, e.g., Sambrook et al, and references citedtherein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts [Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.],use of overlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

III. Production of the Viral Vector

In one embodiment, the simian adenoviral plasmids (or other vectors) areused to produce adenoviral vectors. In one embodiment, the adenoviralvectors are adenoviral particles which are replication-defective. In oneembodiment, the adenoviral particles are rendered replication-defectiveby deletions in the E1a and/or E1b genes. Alternatively, theadenoviruses are rendered replication-defective by another means,optionally while retaining the E1a and/or E1b genes. The adenoviralvectors can also contain other mutations to the adenoviral genome, e.g.,temperature-sensitive mutations or deletions in other genes. In otherembodiments, it is desirable to retain an intact E1a and/or E1b regionin the adenoviral vectors. Such an intact E1 region may be located inits native location in the adenoviral genome or placed in the site of adeletion in the native adenoviral genome (e.g., in the E3 region).

In the construction of useful simian adenovirus vectors for delivery ofa gene to the human (or other mammalian) cell, a range of adenovirusnucleic acid sequences can be employed in the vectors. For example, allor a portion of the adenovirus delayed early gene E3 may be eliminatedfrom the simian adenovirus sequence which forms a part of therecombinant virus. The function of simian E3 is believed to beirrelevant to the function and production of the recombinant virusparticle. Simian adenovirus vectors may also be constructed having adeletion of at least the ORF6 region of the E4 gene, and more desirablybecause of the redundancy in the function of this region, the entire E4region. Still another vector of this invention contains a deletion inthe delayed early gene E2a. Deletions may also be made in any of thelate genes L1 through L5 of the simian adenovirus genome. Similarly,deletions in the intermediate genes IX and IVa₂ may be useful for somepurposes. Other deletions may be made in the other structural ornon-structural adenovirus genes. The above discussed deletions may beused individually, i.e., an adenovirus sequence for use as describedherein may contain deletions in only a single region. Alternatively,deletions of entire genes or portions thereof effective to destroy theirbiological activity may be used in any combination. For example, in oneexemplary vector, the adenovirus sequence may have deletions of the E1genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 andE3 genes, or of E1, E2a and E4 genes, with or without deletion of E3,and so on. As discussed above, such deletions may be used in combinationwith other mutations, such as temperature-sensitive mutations, toachieve a desired result.

An adenoviral vector lacking any essential adenoviral sequences (e.g.,E1a, E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) may be cultured inthe presence of the missing adenoviral gene products which are requiredfor viral infectivity and propagation of an adenoviral particle. Thesehelper functions may be provided by culturing the adenoviral vector inthe presence of one or more helper constructs (e.g., a plasmid or virus)or a packaging host cell. See, for example, the techniques described forpreparation of a “minimal” human Ad vector in International PatentApplication Publication No. WO 96/13597, published May 9, 1996, andincorporated herein by reference.

1. Helper Viruses

Thus, depending upon the simian adenovirus gene content of the viralvectors employed to carry the minigene, a helper adenovirus ornon-replicating virus fragment may be necessary to provide sufficientsimian adenovirus gene sequences necessary to produce an infectiverecombinant viral particle containing the minigene. Useful helperviruses contain selected adenovirus gene sequences not present in theadenovirus vector construct and/or not expressed by the packaging cellline in which the vector is transfected. In one embodiment, the helpervirus is replication-defective and contains a variety of adenovirusgenes in addition to the sequences described above. Such a helper virusis desirably used in combination with an E1-expressing cell line.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helpervirus may optionally contain a second reporter minigene. A number ofsuch reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the transgene on theadenovirus vector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

2. Complementation Cell Lines

To generate recombinant simian adenoviruses (Ad) deleted in any of thegenes described above, the function of the deleted gene region, ifessential to the replication and infectivity of the virus, must besupplied to the recombinant virus by a helper virus or cell line, i.e.,a complementation or packaging cell line. In many circumstances, a cellline expressing the human E1 can be used to transcomplement the chimp Advector. This is particularly advantageous because, due to the diversitybetween the chimp Ad sequences of the invention and the human AdE1sequences found in currently available packaging cells, the use of thecurrent human E1-containing cells prevents the generation ofreplication-competent adenoviruses during the replication and productionprocess. However, in certain circumstances, it will be desirable toutilize a cell line which expresses the E1 gene products can be utilizedfor production of an E1-deleted simian adenovirus. Such cell lines havebeen described. See, e.g., U.S. Pat. No. 6,083,716.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene from SAdV36 under the transcriptional control of a promoter forexpression in a selected parent cell line. Inducible or constitutivepromoters may be employed for this purpose. Examples of such promotersare described in detail elsewhere in this specification. A parent cellis selected for the generation of a novel cell line expressing anydesired SAdV36 gene. Without limitation, such a parent cell line may beHeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], HEK293, KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75]cells, among others. These cell lines are all available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Other suitable parent cell lines may be obtained fromother sources.

Such E1-expressing cell lines are useful in the generation ofrecombinant simian adenovirus E1 deleted vectors. Additionally, oralternatively, cell lines that express one or more simian adenoviralgene products, e.g., E1a, E1b, E2a, and/or E4 ORF6, can be constructedusing essentially the same procedures are used in the generation ofrecombinant simian viral vectors. Such cell lines can be utilized totranscomplement adenovirus vectors deleted in the essential genes thatencode those products, or to provide helper functions necessary forpackaging of a helper-dependent virus (e.g., adeno-associated virus).The preparation of a host cell involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingconventional techniques. Such techniques include cDNA and genomiccloning, which are well known and are described in Sambrook et al.,cited above, use of overlapping oligonucleotide sequences of theadenovirus genomes, combined with polymerase chain reaction, syntheticmethods, and any other suitable methods which provide the desirednucleotide sequence.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549, WEHI, 3T3,10T1/2, HEK 293 cells or PERC6 (both of which express functionaladenoviral E1) [Fallaux, F J et al, (1998), Hum Gene Ther, 9:1909-1917],Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

3. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, and preferably about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10′³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

The vector may be any vector known in the art or disclosed above,including naked DNA, a plasmid, phage, transposon, cosmids, episomes,viruses, etc. Introduction into the host cell of the vector may beachieved by any means known in the art or as disclosed above, includingtransfection, and infection. One or more of the adenoviral genes may bestably integrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently. The gene products may all beexpressed transiently, on an episome or stably integrated, or some ofthe gene products may be expressed stably while others are expressedtransiently. Furthermore, the promoters for each of the adenoviral genesmay be selected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may be regulatedby a specific physiological state of the organism or cell (i.e., by thedifferentiation state or in replicating or quiescent cells) or byexogenously-added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements into various intermediate plasmids),and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude conventional cloning techniques of cDNA such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence. Standard transfection and co-transfection techniques areemployed, e.g., CaPO₄ precipitation techniques. Other conventionalmethods employed include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

For example, following the construction and assembly of the desiredminigene-containing viral vector, the vector is transfected in vitro inthe presence of a helper virus into the packaging cell line. Homologousrecombination occurs between the helper and the vector sequences, whichpermits the adenovirus-transgene sequences in the vector to bereplicated and packaged into virion capsids, resulting in therecombinant viral vector particles. The current method for producingsuch virus particles is transfection-based. However, the invention isnot limited to such methods.

The resulting recombinant simian adenoviruses are useful in transferringa selected transgene to a selected cell. In in vivo experiments with therecombinant virus grown in the packaging cell lines, the E1-deletedrecombinant simian adenoviral vectors of the invention demonstrateutility in transferring a transgene to a non-simian, preferably a human,cell.

IV. Use of the Recombinant Adenovirus Vectors

The recombinant simian SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44based vectors are useful for gene transfer to a human or non-simianveterinary patient in vitro, ex vivo, and in vivo.

The recombinant adenovirus vectors described herein can be used asexpression vectors for the production of the products encoded by theheterologous genes in vitro. For example, the recombinant adenovirusescontaining a gene inserted into the location of an E1 deletion may betransfected into an E1-expressing cell line as described above.Alternatively, replication-competent adenoviruses may be used in anotherselected cell line. The transfected cells are then cultured in theconventional manner, allowing the recombinant adenovirus to express thegene product from the promoter. The gene product may then be recoveredfrom the culture medium by known conventional methods of proteinisolation and recovery from culture.

A SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 derived recombinantsimian adenoviral vector provides an efficient gene transfer vehiclethat can deliver a selected transgene to a selected host cell in vivo orex vivo even where the organism has neutralizing antibodies to one ormore AAV serotypes. In one embodiment, the rAAV and the cells are mixedex vivo; the infected cells are cultured using conventionalmethodologies; and the transduced cells are re-infused into the patient.These compositions are particularly well suited to gene delivery fortherapeutic purposes and for immunization, including inducing protectiveimmunity.

More commonly, the SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44recombinant adenoviral vectors will be utilized for delivery oftherapeutic or immunogenic molecules, as described below. It will bereadily understood for both applications, that the recombinantadenoviral vectors of the invention are particularly well suited for usein regimens involving repeat delivery of recombinant adenoviral vectors.Such regimens typically involve delivery of a series of viral vectors inwhich the viral capsids are alternated. The viral capsids may be changedfor each subsequent administration, or after a pre-selected number ofadministrations of a particular serotype capsid (e.g., one, two, three,four or more). Thus, a regimen may involve delivery of a rAd with afirst simian capsid, delivery with a rAd with a second simian capsid,and delivery with a third simian capsid. A variety of other regimenswhich use the Ad capsids of the invention alone, in combination with oneanother, or in combination with other adenoviruses (which are preferablyimmunologically non-crossreactive) will be apparent to those of skill inthe art. Optionally, such a regimen may involve administration of rAdwith capsids of other non-human primate adenoviruses, humanadenoviruses, or artificial sequences such as are described herein. Eachphase of the regimen may involve administration of a series ofinjections (or other delivery routes) with a single Ad capsid followedby a series with another capsid from a different Ad source.Alternatively, the SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 vectorsmay be utilized in regimens involving other non-adenoviral-mediateddelivery systems, including other viral systems, non-viral deliverysystems, protein, peptides, and other biologically active molecules.

The following sections will focus on exemplary molecules which may bedelivered via the adenoviral vectors of the invention.

A. Ad-Mediated Delivery of Therapeutic Molecules

In one embodiment, the above-described recombinant vectors areadministered to humans according to published methods for gene therapy.A simian viral vector bearing the selected transgene may be administeredto a patient, preferably suspended in a biologically compatible solutionor pharmaceutically acceptable delivery vehicle. A suitable vehicleincludes sterile saline. Other aqueous and non-aqueous isotonic sterileinjection solutions and aqueous and non-aqueous sterile suspensionsknown to be pharmaceutically acceptable carriers and well known to thoseof skill in the art may be employed for this purpose.

The simian adenoviral vectors are administered in sufficient amounts totransduce the target cells and to provide sufficient levels of genetransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the retina and other intraoculardelivery methods, direct delivery to the liver, inhalation, intranasal,intravenous, intramuscular, intratracheal, subcutaneous, intradermal,rectal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe transgene or the condition. The route of administration primarilywill depend on the nature of the condition being treated.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult human or veterinary dosage of the viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles virus.Dosages will range depending upon the size of the animal and the routeof administration. For example, a suitable human or veterinary dosage(for about an 80 kg animal) for intramuscular injection is in the rangeof about 1×10⁹ to about 5×10¹² particles per mL, for a single site.Optionally, multiple sites of administration may be delivered. Inanother example, a suitable human or veterinary dosage may be in therange of about 1×10¹¹ to about 1×10¹⁵ particles for an oral formulation.One of skill in the art may adjust these doses, depending the route ofadministration, and the therapeutic or vaccinal application for whichthe recombinant vector is employed. The levels of expression of thetransgene, or for an immunogen, the level of circulating antibody, canbe monitored to determine the frequency of dosage administration. Yetother methods for determining the timing of frequency of administrationwill be readily apparent to one of skill in the art.

An optional method step involves the co-administration to the patient,either concurrently with, or before or after administration of the viralvector, of a suitable amount of a short acting immune modulator. Theselected immune modulator is defined herein as an agent capable ofinhibiting the formation of neutralizing antibodies directed against therecombinant vector of this invention or capable of inhibiting cytolyticT lymphocyte (CTL) elimination of the vector. The immune modulator mayinterfere with the interactions between the T helper subsets (T_(H1) orT_(H2)) and B cells to inhibit neutralizing antibody formation.Alternatively, the immune modulator may inhibit the interaction betweenT_(H1) cells and CTLs to reduce the occurrence of CTL elimination of thevector. A variety of useful immune modulators and dosages for use ofsame are disclosed, for example, in Yang et al., J. Virol., 70(9)(September, 1996); International Patent Application Publication No. WO96/12406, published May 2, 1996; and International Patent ApplicationNo. PCT/US96/03035, all incorporated herein by reference.

1. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), transforming growth factor (TGF), platelet-derived growth factor(PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one ofthe transforming growth factor superfamily, including TGF, activins,inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, anyone of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF)family of growth factors, nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliaryneurotrophic factor (CNTF), glial cell line derived neurotrophic factor(GDNF), neurturin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors and, interferons,and, stem cell factor, flk-2/flt3 ligand. Gene products produced by theimmune system are also useful in the invention. These include, withoutlimitation, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineeredimmunoglobulins and MHC molecules. Useful gene products also includecomplement regulatory proteins such as complement regulatory proteins,membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1,CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the low density lipoprotein (LDL)receptor, high density lipoprotein (HDL) receptor, the very low densitylipoprotein (VLDL) receptor, and the scavenger receptor. The inventionalso encompasses gene products such as members of the steroid hormonereceptor superfamily including glucocorticoid receptors and estrogenreceptors, Vitamin D receptors and other nuclear receptors. In addition,useful gene products include transcription factors such as jun, fos,max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD andmyogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3,ATF4, ZFS, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,interferon regulation factor (IRF-1), Wilms tumor protein, ETS-bindingprotein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkheadfamily of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VIII, factor IX, cystathione beta-synthase, branched chainketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionylCoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene are particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce self-directed antibodies. T cellmediated autoimmune diseases include Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

The simian adenoviral vectors of the invention are particularly wellsuited for therapeutic regimens in which multiple adenoviral-mediateddeliveries of transgenes is desired, e.g., in regimens involvingredelivery of the same transgene or in combination regimens involvingdelivery of other transgenes. Such regimens may involve administrationof a SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 simian adenoviralvector, followed by readministration with a vector from the sameserotype adenovirus. Particularly desirable regimens involveadministration of a SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 simianadenoviral vector, in which the source of the adenoviral capsidsequences of the vector delivered in the first administration differsfrom the source of adenoviral capsid sequences of the viral vectorutilized in one or more of the subsequent administrations. For example,a therapeutic regimen involves administration of a SAdV-36, SAdV-42.1,SAdV-42.2 and/or SAdV-44 vector and repeat administration with one ormore adenoviral vectors of the same or different serotypes. In anotherexample, a therapeutic regimen involves administration of an adenoviralvector followed by repeat administration with a SAdV-36, SAdV-42.1,SAdV-42.2 and/or SAdV-44 vector which has a capsid which differs fromthe source of the capsid in the first delivered adenoviral vector, andoptionally further administration with another vector which is the sameor, preferably, differs from the source of the adenoviral capsid of thevector in the prior administration steps. These regimens are not limitedto delivery of adenoviral vectors constructed using the SAdV-36,SAdV-42.1, SAdV-42.2 and/or SAdV-44 simian sequences. Rather, theseregimens can readily utilize vectors other adenoviral sequences,including, without limitation, other simian adenoviral sequences, (e.g.,Pan9 or C68, C1, etc), other non-human primate adenoviral sequences, orhuman adenoviral sequences, in combination with one or more of theSAdV-36, SAdV-42.1, SAdV-42.2 or SAdV-44 vectors. Examples of suchsimian, other non-human primate and human adenoviral serotypes arediscussed elsewhere in this document. Further, these therapeuticregimens may involve either simultaneous or sequential delivery ofSAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 adenoviral vectors incombination with non-adenoviral vectors, non-viral vectors, and/or avariety of other therapeutically useful compounds or molecules. Theinvention is not limited to these therapeutic regimens, a variety ofwhich will be readily apparent to one of skill in the art.

B. Ad-Mediated Delivery of Immunogenic Transgenes

The recombinant SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 vectors mayalso be employed as immunogenic compositions. As used herein, animmunogenic composition is a composition to which a humoral (e.g.,antibody) or cellular (e.g., a cytotoxic T cell) response is mounted toa transgene product delivered by the immunogenic composition followingdelivery to a mammal, and preferably a primate. A recombinant simian Adcan contain in any of its adenovirus sequence deletions a gene encodinga desired immunogen. The simian adenovirus is likely to be better suitedfor use as a live recombinant virus vaccine in different animal speciescompared to an adenovirus of human origin, but is not limited to such ause. The recombinant adenoviruses can be used as prophylactic ortherapeutic vaccines against any pathogen for which the antigen(s)crucial for induction of an immune response and able to limit the spreadof the pathogen has been identified and for which the cDNA is available.

Such vaccinal (or other immunogenic) compositions are formulated in asuitable delivery vehicle, as described above. Generally, doses for theimmunogenic compositions are in the range defined above for therapeuticcompositions. The levels of immunity of the selected gene can bemonitored to determine the need, if any, for boosters. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

Optionally, a vaccinal composition of the invention may be formulated tocontain other components, including, e.g., adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art. Examples of suitable adjuvantsinclude, without limitation, liposomes, alum, monophosphoryl lipid A,and any biologically active factor, such as cytokine, an interleukin, achemokine, a ligands, and optimally combinations thereof. Certain ofthese biologically active factors can be expressed in vivo, e.g., via aplasmid or viral vector. For example, such an adjuvant can beadministered with a priming DNA vaccine encoding an antigen to enhancethe antigen-specific immune response compared with the immune responsegenerated upon priming with a DNA vaccine encoding the antigen only.

The recombinant adenoviruses are administered in a “an immunogenicamount”, that is, an amount of recombinant adenovirus that is effectivein a route of administration to transfect the desired cells and providesufficient levels of expression of the selected gene to induce an immuneresponse. Where protective immunity is provided, the recombinantadenoviruses are considered to be vaccine compositions useful inpreventing infection and/or recurrent disease.

Alternatively, or in addition, the vectors of the invention may containa transgene encoding a peptide, polypeptide or protein which induces animmune response to a selected immunogen. The recombinant SAdV-36,SAdV-42.1, SAdV-42.2 and SAdV-44 vectors are expected to be highlyefficacious at inducing cytolytic T cells and antibodies to the insertedheterologous antigenic protein expressed by the vector.

For example, immunogens may be selected from a variety of viralfamilies. Example of viral families against which an immune responsewould be desirable include, the picornavirus family, which includes thegenera rhinoviruses, which are responsible for about 50% of cases of thecommon cold; the genera enteroviruses, which include polioviruses,coxsackieviruses, echoviruses, and human enteroviruses such as hepatitisA virus; and the genera apthoviruses, which are responsible for foot andmouth diseases, primarily in non-human animals. Within the picornavirusfamily of viruses, target antigens include the VP1, VP2, VP3, VP4, andVPG. Another viral family includes the calcivirus family, whichencompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C or the coronavirus family, which includes a number ofnon-human viruses such as infectious bronchitis virus (poultry), porcinetransmissible gastroenteric virus (pig), porcine hemagglutinatingencephalomyelitis virus (pig), feline infectious peritonitis virus(cats), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon-A, B or C hepatitis. Within the coronavirus family, target antigensinclude the E1 (also called M or matrix protein), E2 (also called S orSpike protein), E3 (also called HE or hemagglutin-elterose) glycoprotein(not present in all coronaviruses), or N (nucleocapsid). Still otherantigens may be targeted against the rhabdovirus family, which includesthe genera vesiculovirus (e.g., Vesicular Stomatitis Virus), and thegeneral lyssavirus (e.g., rabies).

Within the rhabdovirus family, suitable antigens may be derived from theG protein or the N protein. The family filoviridae, which includeshemorrhagic fever viruses such as Marburg and Ebola virus, may be asuitable source of antigens. The paramyxovirus family includesparainfluenza Virus Type 1, parainfluenza Virus Type 3, bovineparainfluenza Virus Type 3, rubulavirus (mumps virus), parainfluenzaVirus Type 2, parainfluenza virus Type 4, Newcastle disease virus(chickens), rinderpest, morbillivirus, which includes measles and caninedistemper, and pneumovirus, which includes respiratory syncytial virus.The influenza virus is classified within the family orthomyxovirus andis a suitable source of antigen (e.g., the HA protein, the N1 protein).The bunyavirus family includes the genera bunyavirus (Californiaencephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus(puremala is a hemahagin fever virus), nairovirus (Nairobi sheepdisease) and various unassigned bungaviruses. The arenavirus familyprovides a source of antigens against LCM and Lassa fever virus. Thereovirus family includes the genera reovirus, rotavirus (which causesacute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. No. 5,891,994 and U.S.Pat. No. 6,193,981.] See, also, the HIV and SIV proteins described in D.H. Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R.Amara, et al, Science, 292:69-74 (6 Apr. 2001). In another example, theHIV and/or SIV immunogenic proteins or peptides may be used to formfusion proteins or other immunogenic molecules. See, e.g., the HIV-1 Tatand/or Nef fusion proteins and immunization regimens described in WO01/54719, published Aug. 2, 2001, and WO 99/16884, published Apr. 8,1999. The invention is not limited to the HIV and/or SIV immunogenicproteins or peptides described herein. In addition, a variety ofmodifications to these proteins have been described or could readily bemade by one of skill in the art. See, e.g., the modified gag proteinthat is described in U.S. Pat. No. 5,972,596. Further, any desired HIVand/or SIV immunogens may be delivered alone or in combination. Suchcombinations may include expression from a single vector or frommultiple vectors. Optionally, another combination may involve deliveryof one or more expressed immunogens with delivery of one or more of theimmunogens in protein form. Such combinations are discussed in moredetail below.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus family feline parvovirus (feline enteritis),feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.The herpesvirus family includes the sub-family alphaherpesvirinae, whichencompasses the genera simplexvirus (HSVI, HSVII), varicellovirus(pseudorabies, varicella zoster) and the sub-family betaherpesvirinae,which includes the genera cytomegalovirus (HCMV, muromegalovirus) andthe sub-family gammaherpesvirinae, which includes the generalymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxvirinae, which encompasses the generaorthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus,avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and thesub-family entomopoxvirinae. The hepadnavirus family includes theHepatitis B virus. One unclassified virus which may be suitable sourceof antigens is the Hepatitis delta virus. Still other viral sources mayinclude avian infectious bursal disease virus and porcine respiratoryand reproductive syndrome virus. The alphavirus family includes equinearteritis virus and various Encephalitis viruses.

Immunogens which are useful to immunize a human or non-human animalagainst other pathogens include, e.g., bacteria, fungi, parasiticmicroorganisms or multicellular parasites which infect human andnon-human vertebrates, or from a cancer cell or tumor cell. Examples ofbacterial pathogens include pathogenic gram-positive cocci includepneumococci; staphylococci; and streptococci. Pathogenic gram-negativecocci include meningococcus; gonococcus. Pathogenic entericgram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella;Franisella tularensis (which causes tularemia); yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoans and helminths and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44vectors to deliver immunogens against the variable region of the T cellsis anticipated to elicit an immune response including CTLs to eliminatethose T cells. In RA, several specific variable regions of TCRs whichare involved in the disease have been characterized. These TCRs includeV-3, V-14, V-17 and Vα-17. Thus, delivery of a nucleic acid sequencethat encodes at least one of these polypeptides will elicit an immuneresponse that will target T cells involved in RA. In MS, severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-7 and Vα-10. Thus, delivery ofa nucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12.Thus, delivery of a recombinant simian adenovirus that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in scleroderma.

C. Ad-Mediated Delivery Methods

The therapeutic levels, or levels of immunity, of the selected gene canbe monitored to determine the need, if any, for boosters. Following anassessment of CD8+ T cell response, or optionally, antibody titers, inthe serum, optional booster immunizations may be desired. Optionally,the recombinant SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 vectors maybe delivered in a single administration or in various combinationregimens, e.g., in combination with a regimen or course of treatmentinvolving other active ingredients or in a prime-boost regimen. Avariety of such regimens have been described in the art and may bereadily selected.

For example, prime-boost regimens may involve the administration of aDNA (e.g., plasmid) based vector to prime the immune system to second,booster, administration with a traditional antigen, such as a protein ora recombinant virus carrying the sequences encoding such an antigen.See, e.g., WO 00/11140, published Mar. 2, 2000, incorporated byreference. Alternatively, an immunization regimen may involve theadministration of a recombinant SAdV-36, SAdV-42.1, SAdV-42.2 and/orSAdV-44 vector to boost the immune response to a vector (either viral orDNA-based) carrying an antigen, or a protein. In still anotheralternative, an immunization regimen involves administration of aprotein followed by booster with a vector encoding the antigen.

In one embodiment, a method of priming and boosting an immune responseto a selected antigen by delivering a plasmid DNA vector carrying saidantigen, followed by boosting with a recombinant SAdV-36, SAdV-42.1,SAdV-42.2 and/or SAdV-44 vector is described. In one embodiment, theprime-boost regimen involves the expression of multiproteins from theprime and/or the boost vehicle. See, e.g., R. R. Amara, Science,292:69-74 (6 Apr. 2001) which describes a multiprotein regimen forexpression of protein subunits useful for generating an immune responseagainst HIV and SIV. For example, a DNA prime may deliver the Gag, Pol,Vif, VPX and Vpr and Env, Tat, and Rev from a single transcript.Alternatively, the SIV Gag, Pol and HIV-1 Env is delivered in arecombinant SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 adenovirusconstruct. Still other regimens are described in WO 99/16884 and WO01/54719.

However, the prime-boost regimens are not limited to immunization forHIV or to delivery of these antigens. For example, priming may involvedelivering with a first SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44vector followed by boosting with a second Ad vector, or with acomposition containing the antigen itself in protein form. In oneexample, the prime-boost regimen can provide a protective immuneresponse to the virus, bacteria or other organism from which the antigenis derived. In another embodiment, the prime-boost regimen provides atherapeutic effect that can be measured using convention assays fordetection of the presence of the condition for which therapy is beingadministered.

The priming composition may be administered at various sites in the bodyin a dose dependent manner, which depends on the antigen to which thedesired immune response is being targeted. The amount or situs ofinjection(s) or to pharmaceutical carrier is not a limitation. Rather,the regimen may involve a priming and/or boosting step, each of whichmay include a single dose or dosage that is administered hourly, daily,weekly or monthly, or yearly. As an example, the mammals may receive oneor two doses containing between about 10 μg to about 50 μg of plasmid incarrier. A desirable amount of a DNA composition ranges between about 1μg to about 10,000 μg of the DNA vector. Dosages may vary from about 1μg to 1000 μg DNA per kg of subject body weight. The amount or site ofdelivery is desirably selected based upon the identity and condition ofthe mammal.

The dosage unit of the vector suitable for delivery of the antigen tothe mammal is described herein. The vector is prepared foradministration by being suspended or dissolved in a pharmaceutically orphysiologically acceptable carrier such as isotonic saline; isotonicsalts solution or other formulations that will be apparent to thoseskilled in such administration. The appropriate carrier will be evidentto those skilled in the art and will depend in large part upon the routeof administration. The compositions described herein may be administeredto a mammal according to the routes described above, in a sustainedrelease formulation using a biodegradable biocompatible polymer, or byon-site delivery using micelles, gels and liposomes. Optionally, thepriming step also includes administering with the priming composition, asuitable amount of an adjuvant, such as are defined herein.

Preferably, a boosting composition is administered about 2 to about 27weeks after administering the priming composition to the mammaliansubject. The administration of the boosting composition is accomplishedusing an effective amount of a boosting composition containing orcapable of delivering the same antigen as administered by the primingDNA vaccine. The boosting composition may be composed of a recombinantviral vector derived from the same viral source (e.g., adenoviralsequences of the invention) or from another source. Alternatively, the“boosting composition” can be a composition containing the same antigenas encoded in the priming DNA vaccine, but in the form of a protein orpeptide, which composition induces an immune response in the host. Inanother embodiment, the boosting composition contains a DNA sequenceencoding the antigen under the control of a regulatory sequencedirecting its expression in a mammalian cell, e.g., vectors such aswell-known bacterial or viral vectors. The primary requirements of theboosting composition are that the antigen of the composition is the sameantigen, or a cross-reactive antigen, as that encoded by the primingcomposition.

In another embodiment, the SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44vectors are also well suited for use in a variety of other immunizationand therapeutic regimens. Such regimens may involve delivery of SAdV-36,SAdV-42.1, SAdV-42.2 and/or SAdV-44 vectors simultaneously orsequentially with Ad vectors of different serotype capsids, regimens inwhich SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 vectors are deliveredsimultaneously or sequentially with non-Ad vectors, and regimens inwhich the SAdV-36, SAdV-42.1, SAdV-42.2 and/or SAdV-44 vectors aredelivered simultaneously or sequentially with proteins, peptides, and/orother biologically useful therapeutic or immunogenic compounds. Suchuses will be readily apparent to one of skill in the art.

The following examples illustrate the cloning of SAdV-36, SAdV-42.1,SAdV-42.2 and SAdV-44 and the construction of exemplary recombinantvectors. These examples are illustrative only, and do not limit thescope of the present invention.

Example 1—Isolation of Simian Adenovirus and PCR Analysis

A. Simian Adenovirus 36

Stool samples were recovered from the floors of the Michael E. KeelingCenter for Comparative Medicine and Research, University of Texas M. D.Anderson Cancer Center) facility that houses the chimpanzees (Pantroglodytes) and were frozen and sent to University of Pennsylvania. Thesamples were thawed and suspended in Hanks' Balanced Salt solution, theparticulates pelleted by centrifugation, and sterile filtered through0.2 micron syringe filters. 100 id of each filtered sample wasinoculated into A549 cells grown in Ham's F12 with 10% FBS, 1%Penn-Strep and 50 μg/ml gentamicin. After about 1 to 2 weeks in culture,visual cytopathic effect (CPE) was obvious in cell cultures with severalof the inocula. The presence of adenoviruses in the cultures wasconfirmed by PCR amplification of an internal 1.9 kb of the hexon—theregion encompassing the hypervariable (HVR) regions and that ispredominantly responsible for conferring serotype specificity. Theprimer pair that was utilized for PCR was CAGGATGCTTCGGAGTACCTGAG (SEQID NO:126) and TTGGCNGGDATDGGGTAVAGCATGTT (SEQ ID NO:127). The sequenceobtained from this region was used to make an initial determination ofadenoviral species and novelty of the serotype. The sequence wasdetermined to be in adenovirus subgroup E.

Adenoviral isolates that were determined to be novel were plaquepurified on A549 cells, propagated to high titer and purified on cesiumchloride gradients using standard procedures. Viral DNAs obtained frompurified virus preparations were completely sequenced (Qiagen GenomicsServices, Hilden, Germany).

A total of 25% (36/142) of the M.D. Anderson samples demonstratedcytopathology which in each case was confirmed by PCR to be due to theoutgrowth of at least one adenovirus; the assay is based onamplification of a portion of the hexon gene that spans hypervariableregions 1 to 6 [L. Crawford-Miksza, et al, J Virol 70, 1836 (March1996)].

B. Simian Adenovirus 42.1 (Previously Named SAdV-42)

Stool samples were recovered from the floors of the San Diego zoo thathouses bonobo apes [Pan paniscus)], were frozen and sent to Universityof Pennsylvania. The samples were thawed and suspended in Hanks'Balanced Salt solution, the particulates pelleted by centrifugation, andsterile filtered through 0.2 micron syringe filters. 100 μl of eachfiltered sample was inoculated into A549 cells grown in Ham's F12 with10% FBS, 1% Penn-Strep and 50 μg/ml gentamicin. After about 1 to 2 weeksin culture, visual cytopathic effect (CPE) was obvious in cell cultureswith several of the inocula. The presence of adenoviruses in thecultures was confirmed by PCR amplification of an internal 1.9 kb of thehexon—the region encompassing the hypervariable (HVR) regions and thatis predominantly responsible for conferring serotype specificity. Theprimer pair that was utilized for PCR was CAGGATGCTTCGGAGTACCTGAG (SEQID NO:126) and TTGGCNGGDATDGGGTAVAGCATGTT (SEQ ID NO:127). The sequenceobtained from this region was used to make an initial determination ofadenoviral species and novelty of the serotype. This isolate wasdetermined to be in adenovirus subgroup C.

C. Simian Adenovirus 42.2 (Previously Named SAdV-43)

Stool samples were recovered from the floors of the Jacksonville, Fla.zoo that houses bonobo apes [Pan paniscus)], were frozen and sent toUniversity of Pennsylvania. The samples were thawed and suspended inHanks' Balanced Salt solution, the particulates pelleted bycentrifugation, and sterile filtered through 0.2 micron syringe filters.100 μl of each filtered sample was inoculated into A549 cells grown inHam's F12 with 10% FBS, 1% Penn-Strep and 50 μg/ml gentamicin. Afterabout 1 to 2 weeks in culture, visual cytopathic effect (CPE) wasobvious in cell cultures with several of the inocula. The presence ofadenoviruses in the cultures was confirmed by PCR amplification of aninternal 1.9 kb of the hexon—the region encompassing the hypervariable(HVR) regions and that is predominantly responsible for conferringserotype specificity. The primer pair that was utilized for PCR wasCAGGATGCTTCGGAGTACCTGAG (SEQ ID NO:126) and TTGGCNGGDATDGGGTAVAGCATGTT(SEQ ID NO:127), where “N” represents any amino acid. The sequenceobtained from this region was used to make an initial determination ofadenoviral species and novelty of the serotype. This isolate wasdetermined to be in adenovirus subgroup C.

D. Simian Adenovirus 44

Stool samples were recovered from the floors of the Jacksonville, Fla.zoo that houses bonobo apes [Pan paniscus)], were frozen and sent toUniversity of Pennsylvania. The samples were thawed and suspended inHanks' Balanced Salt solution, the particulates pelleted bycentrifugation, and sterile filtered through 0.2 micron syringe filters.100 μl of each filtered sample was inoculated into A549 cells grown inHam's F12 with 10% FBS, 1% Penn-Strep and 50 μg/ml gentamicin. Afterabout 1 to 2 weeks in culture, visual cytopathic effect (CPE) wasobvious in cell cultures with several of the inocula. The presence ofadenoviruses in the cultures was confirmed by PCR amplification of aninternal 1.9 kb of the hexon—the region encompassing the hypervariable(HVR) regions and that is predominantly responsible for conferringserotype specificity. The primer pair that was utilized for PCR wasCAGGATGCTTCGGAGTACCTGAG (SEQ ID NO:126) and TTGGCNGGDATDGGGTAVAGCATGTT(SEQ ID NO:127), where “N” represents any amino acid. The sequenceobtained from this region was used to make an initial determination ofadenoviral species and novelty of the serotype. This isolate wasdetermined to be in adenovirus subgroup C.

Example 2—Neutralizing Antibody (NAB) in Human Serum to AdenoviralIsolates

Serum samples from 50 normal human subjects (obtained from a blood bank)were tested for their ability to neutralize the ape adenovirus isolates.The prevalence and magnitude of human serum neutralizing antibody (NAB)responses to SAdV-36, SAdV-42.2 and SAdV-44 was determined.

Serum from each of the human subjects was serially diluted and the NABtiter for each of SAdV-36, SAdV-42.2 and SAdV-44 was determined. Thefrequency of seropositivity in samples having an NAB titer greater thanor equal to 1/20 was determined for SAdV-36, SAdV-42.2, SAdV-44, alongwith controls SAdV-24 and HAdV-5. 10% and 28% of samples (NAB greaterthan or equal to 1/20), were positive for SAdV-24 and HAdV-5,respectively. 82%, 97% and 68% of samples (NAB greater than or equal to1/20) were positive for SAdV-36, SAdV-42.2 and SAdV-44, respectively.

Neutralization titers for samples having a NAB titer greater than orequal to 1/20 were plotted as the reciprocal of serum dilution,reflecting the breadth of the response and its distribution for thatsubset of samples. The median neutralizing antibody titers for SAdV-36,42.2, and 44 were found to be more comparable to SAdV-24 than HAdV-5,i.e., they avoid pre-existing immunity in human serum.

Example 3—Vector Construction

A. SAdV-36 (subgroup E)

An E1 deleted vector using the SAdV-36 (species E) DNA (SEQ ID NO:1) wasprepared as described.

(i) Construction of pSR6

A linker containing SmaI, AscI, AvrII, EcoRV sites flanked by PacI sitesis cloned into pBR322 cut with EcoRI and NdeI as follows. The oligomers

pSR6 top (SEQ ID NO: 128)AATTTTAATTAACCCGGGTATCGGCGCGCCTTAACCTAGGGATAGATAT CTTAATTAA and pSR6 bot(SEQ ID NO: 129) TATTAATTAAGATATCTATCCCTAGGTTAAGGCGCGCCGATACCCGGGTTAATTAAwere annealed together to create the linker.

(ii) Cloning of the Viral Left End to the AscI Site (7959)

The viral DNA was digested with AscI and the 7958 bp left end fragmentwas cloned into pSR6 (prepared as in step (i)) digested with SmaI andAscI to yield pSR6 C36 LE.

(iii) E1 Functional Deletion and Insertion of I-CeuI and PI-SceI Sites

The plasmid pSR6 C36 LE (prepared as in step (ii)) was digested withSnaBI and NdeI; the NdeI site was filled in with Klenow. The EcoRVfragment from pBleuSK I-PI was ligated in to create pSR5 C36 LE IP. Thisstep causes a deletion of the SAdV-36 E1 genes. In place of the E1deletion, the fragment that was ligated in harbors recognition sites forthe extremely rare cutter restriction enzymes I-CeuI and PI-SceIrespectively. This allows the subsequent insertion of transgenecassettes in place of the E1 deletion.

(iv) Cloning of the Viral Right End from the XbaI Site (30009)

The plasmid pSR6 C36 LE IP (prepared as in step (iii)) was digested withXbaI and EcoRV. The 6548 bp right end (XbaI digest) fragment from theSAdV-36 DNA was ligated in to create pC36 LE IP RE.

(v) Cloning of the Viral Middle XbaI Fragment (6040-30009)

The plasmid p36 LE IP RE (prepared as in step (iv)) was digested withXbaI. The 23969 bp fragment from the SAdV-36 DNA was ligated in tocreate pC36 IP.

B. SAdV-42.1 (Subgroup C)

An E1 deleted vector using the SAdV-42.1 DNA (SEQ ID NO:33) may beprepared in the same manner as an SAdV-42.2 E1 deleted vector asdescribed in part “C”, below, or by the following method.

An E1 deleted vector using the SAdV-42.1 DNA (SEQ ID NO:33) may beprepared using methods which have been previously described. A linkercontaining SmaI, ClaI, XbaI, SpeI, EcoRV sites flanked by SwaI is clonedinto pBR322 cut with EcoRI and NdeI (pSR5). Viral DNA is digested withXbaI and the 6 kb fragments (left and right ends) are gel purified andligated into pSR5 digested with SmaI and XbaI. 12 minipreps arediagnosed with SmaI and assessed for expected fragment sizes. Miniprepsare sequenced to check the integrity of the viral DNA end. The sequenceobtained is used to correct the left end Qiagen sequence and deduce thecorrect right ITR sequence as well. The plasmid is digested with SnaBIand NdeI and the NdeI site is filled in with Klenow. The EcoRV fragmentfrom pBleuSK I-PI is ligated in. Minipreps are diagnosed using PstI. Theresulting plasmid is digested with XbaI and EcoRV. The right end (XbaIdigest) fragment from the SAdV-42.1 DNA is ligated in. Minipreps arediagnosed using ApaLI. The resulting plasmid is then digested with XbaIand EcoRV. The left end (XbaI digest) fragment from the SAdV-42.1 DNA isligated in and minipreps are diagnosed using MfeI. 293 cells are thentransfected using manufacturer's protocol.

C. SAdV-42.2 (Subgroup C)

An E1 deleted vector using the SAdV-42.2 (subgroup C) DNA (SEQ ID NO:64)was prepared as described.

(i) Construction of pSR2

A linker containing SnaBI, FseI, MluI, PacI and, EcoRV sites flanked byPmeI sites was cloned into pBR322 cut with EcoRI and NdeI as follows.The oligomers

P6 Top (SEQ ID NO: 130)AATTGTTTAAACTACGTAATTAGGCCGGCCGCGCACGCGTGTCATTAAT TAAGCTAGATATCGTTTAAACand P6 Bot (SEQ ID NO: 131)GTTTAAACGATATCTAGCTTAATTAATGACACGCGTGCGCGGCCGGCCT AATTACGTAGTTTAAACATwere annealed together to create the linker.

(ii) Construction of pSR7

The plasmid pSR2 (prepared as in step (i)) was digested with MluI andthe annealed oligomers

(SEQ ID NO: 132) 951top-CGCGCATATGGATCGATCGCTAGCGATCGATCGAATTC and(SEQ ID NO: 133) 951bot-CGCGGAATTCGATCGATCGCTAGCGATCGATCCATATGwere ligated in. This creates a linker harboring SnaBI, FseI, NdeI,NheI, EcoRI, PacI and EcoRV sites flanked by PmeI sites. In pSR7, thislinker replaces the pBR322 fragment from the EcoRI to the NdeI sites.

(iii) Cloning of the Viral Right End

The 826 bp right end fragment extending from the PacI site was clonedinto pSR7 (prepared as in step (ii)) between the EcoRV and PacI sites,to yield pSR7 42.2 RE.

(iv) Construction of E1 Deleted Left End by PCR

A New England Biolabs (NEB) Phusion™ kit was used with each primer at0.5 μM, dNTP at 0.2 mM each, NEB Phusion™ enzyme, and buffers (assupplied in kit). PCR conditions (for all three reactions) were 98° for30 seconds, and 25 cycles of [98° for 10 s, anneal for 30 seconds, 72°for 15 seconds].

PCR 1—the oligomers

(SEQ ID NO: 134) 42.1-CATCATCAATAATATACCTTATTTTGG and (SEQ ID NO: 135)42.2-CCCAGATTACGTATAAAACGGAGACTTTGACCCwere designed to amplify the first 451 bp of the SAdV-42.2 genomicDNA+10 bp overhang (template—SAdV-42.2 DNA, annealing at 61°):

(SEQ ID NO: 136) CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGTGGGCGGAGCGGGGCGGAGTTGGGAGGCGCGGGGCGGGGCGGCGGCGCGGGGCGGGCCGGGAGGTGTGGCGGAAGTTGAGTTTGTAAGTGTGGCGGATGAGACTTGCTAGCGCCGGATGTGGTAAAAGTGACGTTTTTGGAGTGCGACAACGCCCACGGGAAGTGACATTTTTCCCGCGGTTTTTACCGGATGTCGTAGTGAATTTGGGCGTTACCAAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAAATGGGGAAGTGAAATCTGATTAATTTCGCGTTAGTCATACCGCGTAATATTTGCCGAGGGCCGAGGGACTTTGACCGATTACGTGGAGGAATCGCCCAGGTGTTTTTTGAGGTGAATTTCCGCGTTCCGGGTCAAAGTC TCCGTTTTATACGTAATCTGGG

PCR 2—the oligomers

(SEQ ID NO: 137) 42.3 (CGTTTTATACGTAATCTGGGCAACAGGAGGGGT) and(SEQ ID NO: 138) 42.4 (TCGGTCACATCCAGCATCAC)were designed to amplify a 242 bp fragment containing to the 229 bpfragment of SAdV-42.2 (3223 to 3451 bp of the SAdV-42.2 genome) and a 13bp overhang harboring a SnaBI site.

(SEQ ID NO: 139) CGTTTTATACGTAATCTGGGCAACAGGAGGGGTGTGTTCCTGCCCTATCAATGCAACTTGAGCCACACCAAGGTCTTGCTAGAGCCCGAAAGCATGTCCAAGGTGAACCTGAACGGGGTGTTTGACATGACCCTGAAGATATGGAAGGTGCTGAGGTACGACGAGACCAGGTCTCGGTGCAGGCCCTGCGAGTGCGGGGGCAAGCATATGAGGAACCAGCCTGTGATGCTGGATGTGACCGA

There is a 20 bp sequence overlap between PCR 1 and PCR 2 that allowsfor polymerase chain reaction splicing by overlap extension (PCRSOEing). A SnaBI site is present at the junction of the two PCRfragments for insertion of foreign DNA in place of the functional E1deletion.

PCR 3—To combine the products of PCR 1 and PCR 2, 20 μl of each PCR wererun on a 1% SeaPlaque® gel. The bands were cut out and the agarosemelted at 68°. 5 μl of each melted gel was combined with 190 μl ofwater. 4 μl of the diluted mixture was used as template in a 200 μl PCRreaction using the primers 42.1 and 42.4, annealing at 61°. The expectedproduct size was 685 bp.

(v) Cloning of SAdV-42.2 Left End DNA Fragment Harboring a Functional E1Deletion with Insertion of I-CeuI and PI-SceI Sites to Yield pSR7 C42.2LE delE1 IP

The final PCR product was digested with NdeI and ligated into pSR7 42.2RE (prepared as in step (iii)) cut with SnaBI and NdeI to yield pSR7C42.2 LERE. This plasmid was digested with SnaBI and an EcoRV fragmentfrom pBleuSK I-PI harboring I-CeuI and PI-SceI sites was ligated in, toyield pC42.2 LE delE1 IP.

(vi) Cloning of the SAdV-42 Viral Right End from the EcoRI Site (33952)

The SAdV-42.2 viral DNA was digested with EcoRI and the 3767 bp rightend fragment was cloned into pSR7 C14 LE delE1 IP (prepared as in step(v)) between EcoRI and EcoRV to yield pC42.2IP LE RE.

(vii) Cloning of the SAdV-42.2 Viral Nde I (3416-19843) Fragment

The plasmid pC42.2IP LE RE (prepared as in step (vi)) was digested withNdeI and the 16427 bp viral NdeI fragment was ligated in. The clone wascalled pC42.2 del Mlu Pac.

(viii) Cloning of the SAdV-42.2 Viral MluI (12534)-PacI (37000) Fragment

The plasmid pC42.2 del Mlu Pac (prepared as in step (vii)) was digestedwith MluI and PacI and the 24466 bp viral MluI-PacI fragment was ligatedin. The clone was called pC42.2 IP and is a molecular clone of SAdV-42.2genomic DNA harboring an E1 deletion and I-CeuI and PI-SceI recognitionsites in place of the E1 deletion.

D. SAdV-44 (Subgroup C)

An E1 deleted vector using the SAdV-42.2 (subgroup C) DNA (SEQ ID NO:95) was prepared as described.

(i) Construction of pSR2

A linker containing SnaBI, FseI, MluI, PacI and, EcoRV sites flanked byPmeI sites was cloned into pBR322 cut with EcoRI and NdeI as follows.The oligomers

P6 Top (SEQ ID NO: 130)AATTGTTTAAACTACGTAATTAGGCCGGCCGCGCACGCGTGTCATTAAT TAAGCTAGATATCGTTTAAACand P6 Bot (SEQ ID NO: 131)GTTTAAACGATATCTAGCTTAATTAATGACACGCGTGCGCGGCCGGCCT AATTACGTAGTTTAAACATwere annealed together to create the linker.

(ii) Cloning of the Viral Left End by PCR

The left end (PCR, 480 bp+24 bp extension) was cloned into pSR2 betweenSnaBI and MluI using the following primers.

(SEQ ID NO: 134) forward primer-pCATCATCAATAATATACCTTATTTTGG(SEQ ID NO: 140) reverse primer-GATCACGCGTCATGCATGCATATGAATACACTCCGCGTCAGCTG

The underlined bases in the reverse primer sequence correspond torecognition sites for MluI and NdeI respectively. This plasmid yieldedwas pSR2 C44 LE.

(iii) Cloning of the Viral Right End

The 821 bp right end fragment of SAdV-44 extending from the PacI sitewas cloned into pSR2 C44 LE (prepared as in step (ii)) between the EcoRVand PacI sites, to yield pSR2 44 LERE.

(iv) Cloning of the SAdV-44 Viral NdeI (3413) to MluI (12528) Fragment

The SAdV-44 viral DNA was digested with NdeI and MluI and the 9115 bpfragment was cloned into pSR2 44 LERE (prepared as in step (iii))between NdeI and MluI to yield pSR2 C44 LERE-2.

(v) Cloning of the SAdV-44 Viral MluI (12528)-PacI (36891) Fragment

The plasmid pSR2 C44 LERE-2 (prepared as in step (iv)) was digested withMluI and PacI and the 24363 bp viral MluI-PacI fragment was ligated in.The clone was called pC44 IP and is a molecular clone of SAdV-44 genomicDNA harboring an E1 deletion and I-CeuI and PI-SceI recognition sites inplace of the E1 deletion.

Example 4—Insertion of Influenza A Nucleoprotein Expression Cassette

The nucleotide sequence encoding the H1N1 influenza A virusnucleoprotein (NP) (A/Puerto Rico/8/34/Mount Sinai, GenBank accessionnumber AF389119.1) was codon optimized and completely synthesized(Celtek Genes, Nashville, Tenn.). An expression cassette (approximately2.5 kb) composed of the human cytomegalovirus early promoter, anartificial intron derived from the Promega (Madison, Wis.) plasmid pCI,the codon optimized influenza A NP coding sequence and the bovine growthhormone polyadenylation signal was constructed in a shuttle plasmid.

The expression cassette was cut out with the restriction enzymes I-CeuIand PI-SceI and inserted into the plasmid SAdV-36, SAdV-42.2 and SAdV-44molecular clones obtained as described in Example 3 (above). Theresulting molecular clones (harboring the expression cassette forinfluenza nucleoprotein (NP)) were transfected into the E1trans-complementing cell line HEK 293, and recombinant SAdV-36,SAdV-42.2 and SAdV-44 harboring the NP expression cassette were rescued.Adenovirus was purified by cesium chloride density gradientcentrifugation and the particle titer determined by measuring absorbanceat 260 and 280 nm.

Example 5—T Cell Induction by Recombinant SAdV-36 Expressing Influenza ANucleoprotein (NP)

The protocols contained in Roy, et al. [“Partial protection against H5N1influenza in mice with a single dose of a chimpanzee adenovirus vectorexpressing nucleoprotein”, Vaccine 25:6845-6851 (Aug. 6, 2007)], whichis herein incorporated by reference, were utilized in the followingexperiment.

A. Inoculation of Mice

BALB/c mice (6-8 weeks old) were purchased from Charles RiverLaboratories (Wilmington, Mass.). The chimpanzee adenovirus vectors C36CMV PI FluA NP (prepared as described in Example 4) and the human HAdV-5vector AdH5-FluA NP (prepared consistent with Example 4) were used tovaccinate BALB/c mice (5 mice per group). Mice were sedated withintra-peritoneal ketamine/xylazine, and the total adenovirus dose (1011viral particles per mouse) divided into two 25 μl injections givenintra-muscularly to the hind leg tibialis anterior.

B. Analysis of CD8+ T Lymphocytes from Immunized BALB/c Mice

Vaccinated mice were sacrificed 10 days following immunization and theirand their CD8+ T cells were analyzed by stimulating with theimmunodominant peptide TYQRTRALV (SEQ ID NO:141) of influenzanucleoprotein and staining for the cytokines interferon gamma (IFN-γ),interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). It was ofinterest to determine the subset of the CD8+ T cells that werepolyfunctional, i.e., capable of secreting multiple cytokines such asIL-2 and TNF-α, which may be an indication of the quality of the T cellresponse especially with respect to the formation of a memorypopulation. Splenocytes from 5 mice per group were assayed. The resultsare shown in FIG. 1.

The vector based on SAdV-36 is similar to that based on HAdV-5 in itsability to elicit a T cell response against the vaccine immunogen(influenza A nucleoprotein).

All documents recited above, the Sequence Listing, US Patent ApplicationNos.: 61/067,993, 61/068,027, 61/068,024, and 61/068,069, all filed Mar.4, 2008, International Patent Application No. PCT/US2009/001344, filedMar. 3, 2009, U.S. patent application Ser. No. 12/920,648, filed Sep. 2,2010, and U.S. patent application Ser. No. 13/899,037, filed May 21,2013, are incorporated herein by reference. Numerous modifications andvariations are included in the scope of the above-identifiedspecification and are expected to be obvious to one of skill in the art.Such modifications and alterations to the compositions and processes,such as selections of different minigenes or selection or dosage of thevectors or immune modulators are believed to be within the scope of theclaims appended hereto.

1. A recombinant adenovirus having a capsid comprising a hexon protein,a penton protein, and a fiber protein, wherein said hexon protein is thehexon protein of SAdV-44, amino acids 1 to 962 of SEQ ID NO: 105; saidcapsid encapsidating a heterologous nucleic acid molecule carrying agene sequence operably linked to expression control sequences whichdirect transcription, translation, or expression in a host cell.
 2. Theadenovirus according to claim 1, further comprising 5′ and 3′ adenoviruscis-elements necessary for replication and encapsidation.
 3. Theadenovirus according to claim 1, wherein said adenovirus lacks all or apart of the E1 gene.
 4. The adenovirus according to claim 3, whereinsaid adenovirus is replication-defective.
 5. The adenovirus according toclaim 1, wherein said virus has a hybrid capsid.
 6. The adenovirusaccording to claim 5, wherein said capsid comprises a SAdV-42.1,SAdV-42.2 or SAdV-44 fiber protein.
 7. The adenovirus according to claim5, wherein the capsid comprises a SAdV-42.1, SAdV-42.2 or SAdV-44 pentonprotein.
 8. A recombinant adenovirus having a capsid comprising a hexoncontaining a fragment of the simian adenovirus (SAdV) hexon protein anda nucleic acid sequence heterologous to the SAdV, wherein the fragmentof the SAdV hexon protein is the SAdV hexon protein of SEQ ID NO:43, 74or 105 with an N-terminal or C-terminal truncation of about 50 aminoacids in length or is selected from the group consisting of: amino acidresidues 125 to 443 of SEQ ID NO:43, 74 or 105; amino acid residues 138to 441 of SEQ ID NO:43, 74 or 105; amino acid residues 138 to 163 of SEQID NO:43, 74 or 105; amino acid residues 170 to 176 of SEQ ID NO:43, 74or 105; and amino acid residues 404 to 430 of SEQ ID NO:43, 74 or 105.9. The recombinant adenovirus according to claim 8, wherein the capsidfurther comprises a SAdV-42.1, SAdV-42.2 or SAdV-44 fiber protein. 10.The recombinant adenovirus according to claim 8, wherein the capsidfurther comprises a SAdV-42.1, SAdV-42.2 or SAdV-44 penton protein. 11.The recombinant adenovirus according to claim 8, wherein said adenovirusis a pseudotyped adenovirus comprising 5′ and 3′ adenovirus cis-elementsnecessary for replication and encapsidation, said cis-elementscomprising an adenovirus 5′ inverted terminal repeat and an adenovirus3′ inverted terminal repeat.
 12. The recombinant adenovirus according toclaim 8, wherein the adenovirus comprises a nucleic acid sequenceencoding a product operatively linked to sequences which directexpression of said product in a host cell.
 13. The recombinantadenovirus according to claim 8, wherein the recombinant adenoviruscomprises one or more adenovirus genes.
 14. The recombinant adenovirusaccording to claim 8, wherein the recombinant adenovirus isreplication-defective.
 15. The recombinant adenovirus according to claim14, wherein the recombinant adenovirus is deleted in adenovirus E1. 16.A composition comprising an adenovirus according to claim 1 in apharmaceutically acceptable carrier.
 17. A method for targeting a cellhaving an adenoviral receptor comprising delivering to a subject a virusaccording to claim
 1. 18. A vector comprising a simian adenovirus (SAdV)nucleic acid sequence selected from one or more of the group consistingof: (a) 5′ inverted terminal repeat (ITR) sequences; (b) the adenovirusE1a region; (c) the adenovirus E1b region, or a fragment thereofselected from among the group consisting of the open reading frames forthe small T, large T, and IX regions; (d) the E2b region, or a fragmentthereof selected from the group consisting of the open reading framesfor pTP, polymerase, and IVa2; (e) the L1 region, or a fragment thereofselected from among the group consisting of the open reading frames forthe 52/55 kD protein and IIIa protein; (f) the L2 region, or a fragmentthereof selected from the group consisting of the open reading framesfor the penton, VII, V, and pX/Mu proteins; (g) the L3 region, or afragment thereof selected from the group consisting of the open readingframes for the VI, hexon, or endoprotease; (h) the E2a region or theopen reading frame for the DNA binding protein (DBP); (i) the L4 region,or a fragment thereof selected from the group consisting of the openreading frames for the 100 kD protein, the 33 kD homolog, the 22 kDprotein, and VIII; (j) the E3 region, or a fragment thereof selectedfrom the group consisting of the open reading frames for the 12.5Kprotein, CR1-alpha, gp19K, CR1-beta, CR1-gamma, RID-alpha, RID-beta, and14.7K protein; (k) the L5 region, or the open reading frame for thefiber protein; (l) the E4 region, or a fragment thereof selected fromthe group consisting of the open reading frames for the E4 ORF6/7, E4ORF6, E4 ORF4, E4 ORF3, E4 ORF2, and E4 ORF1; and (m) the 3′ ITR; ofSAdV-42.1, SEQ ID NO:33, SAdV-42.2, SEQ ID NO:64, or SAdV-44, SEQ IDNO:95.
 19. A simian adenovirus protein encoded by the vector accordingto claim
 18. 20. A composition comprising an adenovirus according toclaim 1, wherein said adenovirus comprises one or more simian adenovirusproteins selected from the group consisting of: E1a, SEQ ID NO:61, 92 or123; E1b, small T/19K, SEQ ID NO: 54, 65 or 96; E1b, large T/55K, SEQ IDNO: 34, 85 or 116; IX, SEQ ID NO:3 5, 66 or 97; 52/55D, SEQ ID NO: 36,67 or 98; IIIa, SEQ ID NO: 37, 68 or 99; Penton, SEQ ID NO: 38, 69 or100; VII, SEQ ID NO: 39, 70 or 101; V, SEQ ID NO: 40, 71 or 102; pX, SEQID NO: 41, 72 or 103; VI, SEQ ID NO: 42, 73 or 104; Hexon, SEQ ID NO:43, 74 or 105; Endoprotease, SEQ ID NO: 44, 75 or 106; 100 kD, SEQ IDNO: 45, 76 or 107; 33 kD, SEQ ID NO: 63, 94 or 125; 22 kD, SEQ ID NO:56, 87 or 118; VIII, SEQ ID NO: 46, 77 or 108; 12.5 K, SEQ ID NO: 57, 78or 109; CR1-alpha, SEQ ID NO: 47, 88 or 110; gp19K, SEQ ID NO: 48, 79 or111; CR1-beta, SEQ ID NO:49, 80 or 112; CR1-gamma, SEQ ID NO: 58, 89 or119; RID-alpha, SEQ ID NO: 50, 81 or 113; RID-beta, SEQ ID NO: 51, 82 or114; E3/14.7K, SEQ ID NO: 59, 90 or 120; and Fiber, SEQ ID NO: 52, 83 or121.